(19)
JEuropai
Europe*
Office e
Europaisches Patentamt
European Patent Office
uropeen des brevets
■111
(12)
(45) Date of publication and mention
of the grant of the patent:
1 3.1 1 .2002 Bu Met in 2002/46
(21) Application number 98906328.4
(22) Date of filing: 06.02.1998
(H) EP 0 958 495 B1
EUROPEAN PATENT SPECIFICATION
(51) intci7: G01N 27/327, C12Q 1/00
(86) International application number:
PCT/US98/02652
(87) International publication number:
WO 98/035225 (13.08.1998 Gazette 1998/32)
(54) SMALL VOLUME IN VITRO ANALYTE SENSOR
KLEINVOLUMIGER SENSOR 2UR IN-VITRO BESTIMMUNG
DETECTEUR D'UN FAIBLE VOLUME D'ANALYTE IN VITRO
(84) Designated Contracting States:
• TOMASCO, Michael, F.
AT BE CH DE DK ES Fl FR GB GR IE IT LI LU MC
Cupertino, CA 9501 4 (US)
NLPTSE
(74) Representative: Andrae, Steffen, Dr. et al
(30) Priority: 06.02.1997 US 795767
Andrae Flach Haug
Balanstrasse 55
(43) Date of publication of application:
81541 MQnchen (DE)
24.11.1999 Bulletin 1999/47
(56) References cited:
(73) Proprietor Therasense, Inc.
EP-A- 0 255 291 EP-A- 0 286 084
Alameda, CA 94502 (US)
WO-A-95/02817 WO-A-95/1 3534
US-A- 5 120 420 US-A- 5 130 009
(72) Inventors:
US-A- 5 437 999
• HELLER, Adam
Austin, TX 78731 (US)
• C. W. ANDERSON ET AL: "A small volume
• FELDMAN, Benjamin, J.
thin-layer spectroelectrochemical cell for the
Oakland, CA 94618 (US)
study of biological components." ANALYTICAL
• SAY, James
BIOCHEMISTRY, vol. 93, no. 2, 1979, pages
Alameda, CA 94501 (US)
366-372, XP002068329
• VREEKE, Mark, S.
Alameda, CA 94501 (US)
CD
CD
00
to
o
CL
LU
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give
notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in
a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art.
99(1) European Patent Convention).
Printed by Jouw, 75001 PARIS (FR)
EP 0 958 495 B1
Description
Field of the Invention
5 [0001 J This invention relates to analytical sensors for the detection of bioanalytes in a small volume sample.
Background of the Invention
[0002] Analytical sensors are useful in chemistry and medicine to determine the presence and concentration of a
10 biological analyte. Such sensors are needed, for example, to monitor glucose in diabetic patients and lactate during
critical care events. For example, U.S. Pat. No. 5,1 20,420 discloses a biosensor having an insulating base board having
formed thereon, in sequence, leads an electrode system mainly made of carbon, an insulating layer and a reaction
layer composed of an enzyme and an electron acceptor, and being provided thereon with a space defined by a spacer
and a cover.
is [0003] Currently available technology measures bioanalytes in relatively large sample volumes, e.g., generally re-
quiring 3 microliters or more of blood or other biological fluid. These fluid samples are obtained from a patient, for
example, using a needle and syringe, or by lancing a portion of the skin such as the fingertip and "milking" the area to
obtain a useful sample volume. These procedures are inconvenient for the patient, and often painful, particularly when
frequent samples are required. Less painful methods for obtaining a sample are known such as lancing the arm or
20 thigh, which have a lower nerve ending density. However, lancing the body in the preferred regions typically produces
submicroliter samples of blood, because these regions are not heavily supplied with near-surface capillary vessels.
[0004] It would therefore be desirable and very useful to develop a relatively painless, easy to use blood analyte
sensor, capable of performing an accurate and sensitive analysis of the concentration of analytes in a small volume
of sample.
25
Summary of the Invention
[0005] The sensors of the present invention provide a method for the detection and quantification of an analyte in
submicroliter samples. In general, the invention includes a method and sensor for analysis of an analyte in a small
30 volume of sample, preferably by coulometry, as defined in the claims.
Brief Description of the Drawings
[0006] Referring now to the drawings, wherein like reference numerals and letters indicate corresponding structure
35 throughout the several views:
Figure i is a schematic view of a first embodiment of an electrochemical sensor in accordance with the principles
of the present invention having a working electrode and a counter electrode facing each other;
Figure 2 is a schematic view of a second embodiment of an electrochemical sensor in accordance with the principles
40 of the present invention having a working electrode and a counter electrode in a coplanar configuration;
Figure 3 is a schematic view of a third embodiment of an electrochemical sensor in accordance with the principles
of the present invention having a working electrode and a counter electrode facing each other and having an
extended sample chamber;
Figure 4 is a not-to-scale side-sectional drawing of a portion of the sensor of Figures 1 or 3 showing the relative
45 positions of the redox mediator, the sample chamber, and the electrodes;
Figure 5 is a top view of an embodiment of a multiple electrode sensor in accordance with the principles of the
present invention;
Figure 6 is a perspective view of an embodiment of an analyte measurement device in accordance with the prin-
ciples of the present invention having a sample acquisition means and the sensor of Figure 4;
50 Figure 7 is a graph of the charge required to electrooxidize a known quantity of glucose in an electrolyte buffered
solution (filled circles) or serum solution (open circles) using the sensor of Figure 1 with glucose oxidase as the
second electron transfer agent;
Figure 8 is a graph of the average glucose concentrations for the data of Figure 7 (buffered solutions only) with
calibration curves calculated to fit the averages; a linear calibration curve was calculated for the 10-20 mM con-
5 5 centrations and a second order polynomial calibration curve was calculated for the 0-1 0 mM concentrations;
Figure 9 is a Clarke-type clinical grid analyzing the clinical relevance of the glucose measurements of Figure 7;
Figure 1 0 Is a graph of the charge required to electrooxidize a known quantity of glucose in an electrolyte buffered
solution using the sensor of Figure 1 with glucose dehydrogenase as the second electron transfer agent;
2
EP 0 958 495 B1
Figures 11 A, 11 B, and 11 C are top views of three embodiments of an electrochemical sensor of the present in-
vention;
Figures 1 2A and 1 2B are cross-sectional views of another embodiment of an electrochemical sensor of the present
invention formed using a recess of a base material;
Figures 13A and 13B are cross-sectional views of yet another embodiment of an electrochemical sensor or the
present invention formed in a recess of a base material; and
Figures 14A and 14B are cross-sectional view of a further embodiment of an electrochemical sensor of the present
invention formed using a recess of a base material and a sorbent material.,
Detailed Description of the Preferred Embodiment
[0007] When used herein, the following definitions define the stated term:
[0008] An "air-oxidizable mediator" is a redox mediator that is oxidized by air, preferably so that at least 90% of the
mediator is in an oxidized state upon storage in air within a useful period of time, e.g., one month or less, and, preferably,
one week or less, and, more preferably, one day or less.
[0009J A "biological fluid" is any body fluid in which the analyte can be measured, for example, blood, interstitial fluid,
dermal fluid, sweat, and tears.
[0010] The term "blood" in the context of the invention includes whole blood and its cell-free components, namely,
plasma and serum.
[001 1] "Coulometry" is the determination of charge passed or projected to pass during complete or nearly complete
electrolysis of the analyte, either directly on the electrode or through one or more electron transfer agents. The charge
is determined by measurement of charge passed during partial or nearly complete electrolysis of the analyte or, more
often, by multiple measurements during the electrolysis of a decaying current and elapsed time. The decaying current
results from the decline in the concentration of the electrolyzed species caused by the electrolysis.
[0012] A "counter electrode" refers to an electrode paired with the working electrode, through which passes an elec-
trochemical current equal in magnitude and opposite in sign to the current passed through the working electrode, in
the context of the invention, the term "counter electrode" is meant to include counter electrodes which also function
as reference electrodes (i.e. a counter/reference electrode).
[0013] An "electrochemical sensor" is a device configured to detect the presence and/or measure the concentration
of an analyte via electrochemical oxidation and reduction reactions on the sensor. These reactions are transduced to
an electrical signal that can be correlated to an amount or concentration of analyte.
[0014] "Electrolysis" is the electrooxidation or electroreduction of a compound either directly at an electrode or via
one or more electron transfer agents.
[0015] The term "facing electrodes" refers to a configuration of the working and counter electrodes in which the
working surface of the working electrode is disposed in approximate opposition to a surface of the counter electrode
and where the distance between the working and counter electrodes is less than the width of the working surface of
the working electrode.
[0016] A compound is "immobilized" on a surface when it is entrapped on or chemically bound to the surface.
[0017] The "measurement zone" is defined herein as a region of the sample chamber sized to contain only that
portion of the sample that is to be interrogated during the analyte assay.
[0018] A "non-leachable" or "non-releasable" compound is a compound which does not substantially diffuse away
from the working surface of the working electrode for the duration of the analyte assay..
[0019] A "redox mediator" is an electron transfer agent for carrying electrons between the analyte and the working
electrode, either directly, or via a second electron transfer agent.
[0020] A "second electron transfer agent" is a molecule which carries electrons between the redox mediator and the
analyte: -
[0021 ] "Sorbent material" is material which wicks, retains, or is wetted by a fluid sample in its void volume and which
does not substantially prevent diffusion of the analyte to the electrode.
[0022] A "working electrode" is an electrode at which analyte is electrooxidized or electroreduced with or without the
agency of a redox mediator.
[0023] A "working surface" is that portion of the working electrode which is coated with redox mediator and configured
for exposure to sample.
[0024] The small volume, in vitro analyte sensors of the present Invention are designed to measure the concentration
of an analyte in a portion of a sample having a volume less than about 1 \iL, preferably less than about 0.5 \xL, more
preferably less than about 0.2 jiL, and most preferably less than about 0.1 jiL. The analyte of interest is typically
provided in a solution or biological fluid, such as blood or serum. Referring to the Drawings in general and Figures 1 -
4 in particular, a small volume, in vitro electrochemical sensor 20 of the invention generally includes a working electrode
22, a counter (or counter/reference) electrode 24, and a sample chamber 26 (see Figure 4). The sample chamber 26
3
EP0 958 495 B1
is configured so that when a sample is provided in the chamber the sample is in electrolytic contact with both the
working electrode 22 and the counter electrode 24. This allows electrical current to flow between the electrodes to
effect the electrolysis (electrooxidation or electroreduction) of the anafyte.
5 Working Electrode
[0025] The working electrode 22 may be formed from a molded carbon fiber composite or it may consist of an inert
non-conducting base material, such as polyester, upon which a suitable conducting layer is deposited. The conducting
layer should have relatively low electrical resistance and should be electrochemically inert over the potential range of
10 the sensor during operation. Suitable conductors include gold, carbon, platinum, ruthenium dioxide and palladium, as
well as other non-corroding materials known to those skilled in the art. The electrode and/or conducting layers are
deposited on the surface of the inert material by methods such as vapor deposition or printing.
[0026] A tab 23 may be provided on the end of the working electrode 22 for easy connection of the electrode to
external electronics (not shown) such as a voltage source or current measuring equipment. Other known methods or
15 structures may be used to connect the working electrode 22 to the external electronics.
Sensing Layer and Redox Mediator
[0027] A sensing layer 32 containing a non-leachable (i.e., non-releasable) redox mediator is disposed on a portion
20 of the working electrode 22. Preferably, there is little or no leaching of the redox mediator away from the working
electrode 22 into the sample during the measurement period, which is typically less than about 5 minutes. More pref-
erably, the redox mediators of the present invention are bound or otherwise immobilized on the working electrode 22
to prevent undesirable leaching of the mediator into the sample. A diffusing or leachable (i.e., releasable) redox mediator
is not desirable when the working and counter electrodes are close together (i.e., when the electrodes are separated
25 by less than about 1 mm), because a large background signal is typically produced as the unbound mediator shuttles
electrons between the working and counter electrodes, rather than between the analyte and the working electrode.
This and other problems have hindered the development of low resistance cells and increased the minimum sample
size required for determination of analyte concentration.
[0028] Application of sensing layer 32 on working electrode 22 creates a working surface on that electrode. In general,
30 the working surface is that portion of the working electrode 22 coated with mediator and able to contact a fluid sample.
If a portion of the sensing layer 32 is covered by a dielectric or other material, then the working surface will only be
that portion of the electrode covered by redox mediator and exposed for contact with the sample.
[0029] The redox mediator mediates a current between the working electrode 22 and the analyte and enables the
electrochemical analysis of molecules which are not suited for direct electrochemical reaction on an electrode. The
35 mediator functions as an electron transfer agent between the electrode and the analyte.
[0030] Almost any organic or organometallic redox species can be used as a redox mediator. In general, the preferred
redox mediators are rapidly reducible and oxidizable molecules having redox potentials a few hundred millivolts above
or below that of the standard calomel electrode (SCE), and typically not more reducing than about -100 mV and not
more oxidizing than about +400mV versus SCE. Examples of organic redox species are quinones and quinhydrones
40 and species that in their oxidized state have quinoid structures, such as Nile blue and indophenol. Unfortunately, some
quinones and partially oxidized quinhydrones react with functional groups of proteins such as the thiol groups of
cysteine, the amine groups of lysine and arginine, and the phenolic groups of tyrosine which may render those redox
species unsuitable for some of the sensors of the present invention, e.g., sensors that will be used to measure analyte
in biological fluids such as blood.
45 [0031] In general, mediators suitable for use in the invention have structures which prevent or substantially reduce
the diffusional loss of redox species during the period of time that the sample is being analyzed. The preferred redox
mediators include a redox species bound to a polymer which can in turn be immobilized on the working electrode.
Useful redox mediators and methods for producing them are described in U.S. Patent Nos. 5,264,104; 5,356,786;
5,262,035; and 5,320,725, herein incorporated by reference. Although, any organic or organometallic redox species
so can be bound to a polymer and used as a redox mediator, the preferred redox species is a transition metal compound
or complex. The preferred transition metal compounds or complexes Include osmium, ruthenium, iron, and cobalt
compounds or complexes. The most preferred are osmium compounds and complexes.
[0032] One type of non-releasable polymeric redox mediator contains a redox species covalently bound in a poly-
meric composition: An example of this type of mediator is poly(vinylferrocene).
55 [0033] Alternatively, a suitable non-releasable redox mediator contains an ionically-bound redox species. Typically,
these mediators include a charged polymer coupled to an oppositely charged redox species. Examples of this type of
mediator include a negatively charged polymer such as Nation® (Du Pont) coupled to a positively charged redox species
such as an osmium or ruthenium polypyridyl cation. Another example of an ionically-bound mediator is a positively
4
EP 0 958 495 B1
charged polymer such as quaternized poly(4-vinyl pyridine) or po(y(1 -vinyl imidazole) coupled to a negatively charged
redox species such as ferricyanide or ferrocyanide.
[0034] In another embodiment of the invention, the suitable non-releasable redox mediators include a redox species
coordi natively bound to the polymer. For example, the mediator may be formed by coordination of an osmium or cobalt
5 2, 2'-bipyridyl complex to poly(1 -vinyl imidazole) or poly(4-vinyl pyridine).
[0035] The preferred redox mediators are osmium transition metal complexes with one or more ligands having a
nitrogen-containing heterocycle such as 2,2 , -bipyridine, 1,10-phenanthrbline or derivatives thereof. Furthermore, the
preferred redox mediators also have one or more polymeric ligands having at least one nitrogen-containing heterocycle,
such as pyridine, imidazole, or derivatives thereof. These preferred mediators exchange electrons rapidly between
10 each other and the electrodes so that the complex can be rapidly oxidized and reduced.
[0036] In particular, it has been determined that osmium cations complexed with two ligands containing 2,2'-bipyri-
dine, 1 , 1 0-phenanthroline, or derivatives thereof, the two ligands not necessarily being the same, and further complexed
with a polymer having pyridine or imidazole functional groups form particularly useful redox mediators in the small
volume sensors of the present invention. Preferred derivatives of 2,2'-bipyridine for complexation with the osmium
'5 cation are 4,4'-dimethyl-2 t 2'-bipyridine and mono-, di-, and polyalkoxy^^'-bipyridines, such as 4,4 , -dimethoxy-2,2'-
bipyridine, where the carbon to oxygen ratio of the alkoxy groups is sufficient to retain solubility of the transition metal
complex in water. Preferred derivatives of 1 ,1 0-phenanthroline for complexation with the osmium cation are 4,7-dime-
thyl-1,1 0-phenanthroline and mono-,di-, and polyalkoxy-1,10-phenanthrolines, such as 4,7-dimethoxy-1,1 0-phenan-
throline, where the carbon to oxygen ratio of the alkoxy groups is sufficient to retain solubility of the transition metal
20 complex in water. Preferred polymers for complexation with the osmium cation include poly(1 -vinyl imidazole), e.g.,
PVI, and poly(4-vinyl pyridine), e.g., PVP, either alone or with a copolymer. Most preferred are redox mediators with
osmium complexed with poly(1 -vinyl imidazole) alone or with a copolymer.
[0037] The preferred redox mediators have a redox potential between about - 150 mV to about 4400 mV versus the
standard calomel electrode (SCE). Preferably, the potential of the redox mediator is between about -1 00 mV and +1 00
25 mV and more preferably, the potential is between about -50 mV and +50 mV. The most preferred redox mediators have
osmium redox centers and a redox potential more negative than +100 mV versus SCE, more preferably the redox
potential is more negative than +50 mV versus SCE, and most preferably is near -50 mV versus SCE.
[0038] It is also preferred that the redox mediators of the inventive sensors be air-oxidizable. This means that the
redox mediator is oxidized by air, preferably so that at least 90% of the mediator is in an oxidized state prior to intro-
30 duction of sample into the sensor. Air-oxidizable redox mediators include osmium cations complexed with two mono-,
di-, or polyalkoxy-2,2'-bipyridine or mono-, di-, or polyalkoxy-1 ,1 0-phenanthroline ligands, the two ligands not neces-
sarily being the same, and further complexed with polymers having pyridine and imidazole functional groups. In par-
ticular, Os[4,4 , -dimethoxy-2,2'-bipyridine] 2 CI +/+2 complexed with poly(4-vinyl pyridine) or poly(1 -vinyl imidazole) attains
approximately 90% or more oxidation in air.
35 [0039] In a preferred embodiment of the invention, the sensing layer 32 includes a second electron transfer agent
which is capable of transferring electrons to or from the redox mediator and the analyte. One example of a suitable
second electron transfer agent is an enzyme which catalyzes a reaction of the analyte. For example, a glucose oxidase
or glucose dehydrogenase, such.as pyrroloquinoline quinone glucose dehydrogenase (PQQ), is used when the analyte
is glucose. A lactate oxidase fills this role when the analyte is lactate. These enzymes catalyze the electrolysis of an
40 analyte by transferring electrons between the analyte and the electrode via the redox mediator. Preferably, the second
electron transfer agent is non-leachable, and more preferably immobilized on the electrode, to prevent unwanted leach-
ing of the agent into the sample. This is accomplished, for example, by cross linking the second electron transfer agent
with the redox mediator, thereby providing a sensing layer with non-leachable components!
[0040] To prevent electrochemical reactions from occurring on portions of the working electrode not coated by the
45 mediator, a dielectric 40 may be deposited on the electrode over, under, or surrounding the region with the bound redox
mediator, as shown in Figure 4. Suitable dielectric materials include waxes and non-conducting organic polymers such
as polyethylene. Dielectric 40 may also cover a portion of the redox mediator on the electrode. The covered portion of
the mediator will not contact the sample, and, therefore, will not be a part of the electrode's working surface.
po Counter Electrode
[0041] Counter electrode 24 may be constructed in a manner similar to working electrode 22. Counter electrode 24
may also be a counter/reference electrode. Alternatively, a separate reference electrode may be provided in contact
with the sample chamber. Suitable materials for the counter/reference or reference electrode include Ag/AgCI printed
55 on a non-conducting base material or silver chloride on a silver metal base. If the counter electrode is not a reference
electrode, the same materials and methods may be used to make the counter electrode as are available for constructing
the working electrode 22, however, no redox mediator is immobilized on the counter or counter/reference electrode
24. A tab 25 may be provided on the electrode for convenient connection to the external electronics (not shown), such
5
EP 0 958 495 B1
as a coulometer or other measuring device.
MtiZinalZT^T ° f T inVen ! i0n - W ° rki " 9 e ' eC,r0de 22 and C0Unter e,ectrode 24 are *Posed opposite to
and facing each other to form a facing electrode pair as depicted in Figures 1 and 3. In this preferred conjuration
SSir disp rH ed H be T n the ^ e,ectrodi For th,s facina
SSLTllJ2^H!r d n °' bS V? ° PP ° Sin9 e8Ch 0,her> ,h6y may be sli 9 h,, y offeet - furthermore, the two
SSJSSZSSSST 8 S ' Ze - ? erablV ' C ° Un,er ele ° ,r0de 24 iS at least as ,ar 9 e as the worki ng s^ace
0 totMh^
oLff^h L 6 and W ° rkin9 e,eCtr ° de are Wl,nin the ° f the '"^ntion. However, the sep ra
«^
[0044] Figures 11 A, 11B, and 11C illustrate different embodiments of pairs effacing electrodes 22 24 as described
whteh thesr, 21 IT!*' b6,Ween th8 *» e,eCtr ° d6S 22 ' 24 C0 - S P°"* ^ the ^mfasu^n, zone iJ
caoad or 'T^' °' e ' eCtr ° deS ^ 24 is a conducti "9 surfac « and acts as a plate of a
capac tor. The measurement zone between the electrodes 22. 24 acts as a dielectric layer between the plates Thus
e s: s T2 a 24 a t^: 2 ,h : t ele r des 22, 24 - This capaci,ance is a ,unc,ion ° f *• *• £25
22 24 ThT H I" ^ * f^™*" 24 ' Bnd lh6 die,6CtriC COnS,ant of the ma teria. between
of lh ; mil, h t » ' " °' ,He re9 ' Cn 21 °' the overta PPing electrodes 22, 24 and the dielectric constant
1 T h ? n , ,he , e,eCtr ° deS (e g > air ° r a SOrbent material > are know n, then the separation between the
electrodes can be calculated to determine the volume of the measurement zone
S25 JS™ '^ S T eS embodiment of the inventi °" whi=h the electrodes 22. 24 are positioned in a
facing element. For the capacitance to be uniform among.slmllarly constructed analyte sensor having this par
hcular sensor configuration, the registration (i.e., the positioning of the two electrodes relative to one anotner should
the £Zl°T im J"? ° f ,he eleCtr0deS iS Shmed in the x " y P |ane «™ the position shown n Fig ra1 1A
mLremlm zone P ' ' Wi " The Same princi «* e ho,ds ,or »• * the
[0046] figures 1 1 B and 1 1 C illustrate other embodiments of the invention with electrodes 22, 24 in a facina arranae-
distance. ,n the x-y plane relative to the other electrode without a change in the capacitance or the volume™
measurement zone. In these electrode arrangements, each electrode 22 24 includes an arm 122 124 respLSvl
which overlaps with the corresponding arm of theotherelectmde.Thetwoa.rns^
Selc eac"h oZT^'TZ ^ ^ 122 ' 124 " diSp ° Sed 31 8 " ^ 123 « -hichHeater hTn To,
relative to each other. In addrtion, the two arms 122, 124 extend beyond the region 21 of overlap (i e each arm has
oTthe SKStt? 2TT TT nCe ^ len9,h * thS ^ 222 224 > -d « 2
IkLn! ^r >- , ,r ° de arran 9 emente . there can be a certain amount of allowed imprecision in the
EX *.? the electrodes 22. 24 which does not change the capacitance of the electrode arrangement. A desi ed
^3 al which Z 'T"" the ; e 9 is t ra tion can be designed into the electrode arrangement by varying the angle
of fh« r!„on ,1 r T' 1 T 24overi aP and the size ofthe extra length of each arm 122, 124 relative to the width 121
of he reg,on 21 of overlap. Typically, the closer that the arms 122, 124 are to being perpendicular (i e anqle 123 is
90-). the greater the allowed imprecision. Also, the greater the extra length of each a!n?22. 124 (which may bom be
he same ,ength or different lengths) relative to the width 121 of the region 21 of overlap the g^rThe atwed
given elec rode width, thickness, and angle 123 of intersection with the other electrode). Thus, the minimum distance
tST-SSS^T . S ™ ed r la, " e t0 the 0ther is ba,a " ced against the amount of materia, needeZSlec
trodes. Typically, the angle 123 of intersection ranges from 5 to 90 degrees, preferably, 30 to 90 degrees and mere
P^ferably 60 to 90 degrees. Typically, the ratio of the extra length of an arm \ 22, 124 (S^ndinglo the difference
between the arm length 222, 224 and the width 1 21 of the region 21 of overlap) versus the width 21 of the region 2^
of overlap ranges from 0.1 :1 to 50:1, preferably 1 :1 to 15:1 , and more preferabiy 4:1 to 101 '
case, the sample chamber 26 is in contact with both electrodes and is bounded on the side opposKe the electrodes
polyester 9 "" t ^ * **** materia ' S ^ ^ ™" base inc,ude n^onducJng maten^S I
fonmtd 0 n°Sr nf rr ti0 r " ^ T"™ m ate0 pMe ' For exam P'^ the two electrodes may be
haTSm a S TnT^ ™ ™t * °^ °" e SUCh confi 9 ur ation would have the eiectrodes on surfaces
1 1 9 , 9 ,e - Ano,her P° sslble configuration has the electrodes on a curved surface such as the interior of
tube. This ,s another example of a facing electrode pair. Arternatively, the electrodes may be placed near each other
6
EP 0 958 495 B1
on the tube wall (e.g., one on top of the other or side-by-side).
[0049] In any configuration, the two electrodes must be configured so that they do not make direct electrical contact
with each other, to prevent shorting of the electrochemical sensor. This may be difficult to avoid when the facing elec-
trodes having a short (less than about 100u/n) distance between them.
5 [0050] A spacer 28 can be used to keep the electrodes apart when the electrodes face each other as depicted in
Figures 1 and 3. The spacer is typically constructed from an inert non-conducting material such as polyester, Mylar™,
Kevlar™ or any other strong, thin polymer film, or, alternatively, a thin polymer film such as a Teflon™ film, chosen for
its chemical inertness. In addition to preventing contact between the electrodes, the spacer 28 often functions as a
portion of the boundary for the sample chamber 26 as shown in Figures 1-4.
10
Sample Chamber
[0051] The sample chamber 26 is typically defined by a combination of the electrodes 22, 24, an inert base 30, and
a spacer 28 as shown in Figures 1 -4. A measurement zone is contained within this sample chamber and is the region
15 of the sample chamber that contains only that portion of the sample that is interrogated during the analyte assay. In
the embodiment of the invention illustrated in Figures 1 and 2, sample chamber 26 is the space between the two
electrodes 22, 24 and/or the inert base 30. In this embodiment, the sample chamber has a volume that is preferably
less than about 1 ul, more preferably less than about 0.5 u.L, and most preferably less than about 0.2 \iL. In the
embodiment of the invention depicted in Figures 1 and 2, the measurement zone has a volume that is approximately
20 equal to the volume of the sample chamber.
[0052] In another embodiment of the invention, shown in Figure 3, sample chamber 26 includes much more space
than the region proximate electrodes 22, 24. This configuration makes it possible to provide multiple electrodes in
contact with one or more sample chambers, as shown in Figure 5. In this embodiment, sample chamber 26 is preferably
sized to contain a volume of less than about 1 ^iL, more preferably less than about 0.5 \xL, and most preferably less
25 than about 0.2 ul. The measurement zone (i.e., the region containing the volume of sample to be interrogated) is
generally sized to contain a volume of sample of less than about 1 jxL, preferably less than about 0.5 pi, more preferably
less than about 0.2 jxL, and most preferably less than about 0.1 u.L. One particularly useful configuration of this em-
bodimentpositions working electrode 22 and counter electrode 24 facing each other, as shown in Figure 3. In this
embodiment, the measurement zone, corresponding to the region containing the portion of the sample which will be
30 interrogated, is the portion of sample chamber 26 bounded by the working surface of the working electrode and disposed
between the two facing electrodes. When the surface of the working electrode is not entirely covered by redox mediator,
the measurement zone is the space between the two facing electrodes that has a surface area corresponding to the
working surface (i.e., redox mediator-covered surface) of working electrode 22 and a thickness corresponding to the
separation distance between working electrode 22 and counter electrode 24.
35 [0053] In both of the embodiments discussed above, the thickness of the sample chamber and of the measurement
zone correspond typically to the thickness of spacer 28 (e.g., the distance between the electrodes in Figures 1 and 3,
or the distance between the electrodes and the inert base in Figure 2). Preferably, this thickness is small to promote
rapid electrolysis of the analyte, as more of the sample will be in contact with the electrode surface for a given sample
volume. In addition, a thin sample chamber helps to reduce errors from diffusion of analyte into the measurement zone
*o from other portions of the sample chamber during the analyte assay, because diffusion time is long relative to the
measurement time. Typically, the thickness of the sample chamber is less than about 0.2 mm. Preferably, the thickness
of the sample chamber is less than about 0,1 mm and, more preferably, the thickness of the sample chamber is about
0.05 mm or less.
[0054] The sample chamber may be formed by other methods. Exemplary methods include embossing, indenting,
45 or otherwise forming a recess in a substrate within which either the working electrode 22 or counter electrode 24 is
formed. Figures 12A and 12B illustrate one embodiment of this structure. First, a conducting layer 100 is formed on
an inert non-conducting base material 102. As described above, the conducting layer 100 can include gold, carbon,
platinum, ruthenium dioxide, palladium, or other non-corroding materials. The inert non-conducting base material 102
can be made using a polyester, other polymers, or other non-conducting, deformable materials. A recess 104 is then
50 formed in a region of the non-conducting base material 102 so that at least a portion of the conducting layer 100 is
included in the recess 104. The recess 104 may be formed using a variety of techniques including indenting, deforming,
or otherwise pushing in the base material 102. One additional exemplary method for forming the recess includes em-
bossing the base material 102. For example, the base material 102 may be brought into contact with an .embossing
roll or stamp having raised portions, such as punch members or channels, to form the recess 104. In some embodi-
55 ments, the base material 1 02 may be heated to soften the material.
[0055] The recess 104 may be circular, oval, rectangular, or any other regular or irregular shape. Alternatively, the
recess 104 may be formed as a channel which extends along a portion of the base material 102. The conducting layer
100 may extend along the entire channel or only a portion of the channel. The measurement zone may be restricted
7
EP 0 958 495 B1
to a particular region within the channel by, for example, depositing the sensing layer 32 on only that portion of the
conducting layer 100 within the particular region of the channel. Alternatively, the measurement zone may be defined
by placing a second electrode 107 over only the desired region of the first electrode 105.
[0056] At least a portion, and in some cases, all, of the conducting layer 1 00 is situated in the recess 1 04. This portion
5 of the conducting layer 100 may act as a first electrode 1 05 (a counter electrode or, preferably, a working electrode).
If the conducting layer 100 forms the working electrode, then a sensing layer 32 may be formed over a portion of the
conducting layer 100 by depositing the non-leachable mediator and optional second electron transfer agent in the
recess 104, as shown in Figure 12B. A second electrode 107 is then formed by depositing a second conducting layer
on a second base material 106. This second electrode 107 is then positioned over the first electrode 105 in a facing
10 arrangement. Although not illustrated, it will be understood that if the first electrode 1 05 were to function as a counter
electrode, then the sensing layer 32 would be deposited on the second electrode 107 which would then function as
the working electrode.
[0057] In one embodiment, the second base material 1 08 rests on a portion of the first base material 1 02 and/or the
conducing layer 100 which is not depressed, so that the second electrode 107 extends into the recess. In another
15 embodiment, there is a spacer (not shown) between the first and second base materials 1 02, 1 08. In this embodiment,
the second electrode 107 may or may not extend Into the recess. In any case, the first and second electrodes 105,
i07 do not make contact, otherwise the two electrodes would be shorted.
[0058] The depth of the recess 1 04 and the volume of the conductive layer 1 00, sensing layer 32, and the portion,
if any, of the second electrode 107 in the recess 1 04 determines the volume of the measurement zone. Thus, the
20 predictability of the volume of the measurement zone relies on the extent to which the formation of the recess 104 is
uniform.
[0059] In addition to the conducting layer 100, a sorbent layer 103, described in detail below, may be deposited on
the base material 102 prior to forming the recess 104, as shown in Figure 14A. The sorbent material 103 may be
indented, embossed, or otherwise deformed with the conducting layer 1 00 and base material 1 02, as shown in Figure
25 14B. Alternatively, the sorbent material 1 03 may be deposited after the conducting layer 1 00 and base material 1 02
are indented, embossed, or otherwise deformed to make the recess 104.
[0060] In another exemplary method for forming the analyte sensor. A recess 114 in formed in a first base material
112, as shown in Figures 13A and 13B. The recess may be formed by indenting, embossing, etching (e.g., using
photolithographic methods or laser removal of a portion of the base material), or otherwise deforming or removing a
30 portion of the base material 112. Then a first conducting layer 110 is formed in the recess 114. Any of the conductive
materials discussed above may be used. A preferred material is a conductive ink, such as a conductive carbon ink
available, for example, from Ercon, Inc. (Wareham, MA). The conductive ink typically contains metal or carbon dissolved
or dispersed in a solvent or dispersant. When the solvent or dispersant is removed, the metal or carbon forms a con-
ductive layer 110 that can then be used as a first electrode 115. A second electrode 117 can be formed on a second
35 base material 11 6 and positioned over the recess 1 1 4, as described above. In some' embodiments, a sensing layer 32
is formed on the first electrode 115 to form a working electrode, as shown in Figure 13B. In other embodiments, the
sensing layer 32 may be formed on the second electrode 117 to form a working electrode. Furthermore, a sorbent
material (not shown) may be formed within the recess, for example, on the first electrode 115.
[0061] A binder, such as a polyurethane resin, cellulose derivative, elastomer (e.g., silicones, polymeric dienes, or
40 acrylonitrile-butadiene-styrene (ABS) resins), highly fluorinated polymers, or the like, may also be included in the con-
ductive ink. Curing the binder may increase the conductivity of the conductive layer 110, however, curing is not nec-
essary. The method of curing the binder may depend on the nature of the particular binder that is used. Some binders
are cured by heat and/or ultraviolet light.
[0062] These structures allow for the formation of electrochemical sensors in which the volume of the measurement
<5 zone depends, at least in part, on the accuracy and reproducibility of the recess 1 04. Embossing, laser etching, pho-
tolithographic etching and other methods can be used to make reproducible recesses 104, even on the scale of 200
nm or less.
Sorbent Material
50
[0063] The sample chamber may be empty before the sample is placed in the chamber. Alternatively, the sample
chamber may include a sorbent material 34 to sorb and hold a fluid sample during the measurement process. Suitable
sorbent materials include polyester, nylon, cellulose, and cellulose derivatives such as nitrocellulose. The sorbent
material facilitates the uptake of small volume samples by a wicking action which may complement or, preferably,
55 replace any capillary action of the sample chamber.
[0064] In some embodiments, the sorbent material is deposited using a liquid or slurry in which the sorbent material
is dissolved or dispersed. The solvent or dispersant In the liquid or slurry may then be driven off by heating or evaporation
processes. Suitable sorbent materials include, for example, cellulose or nylon powders dissolved or dispersed in a
8
EP 0 958 495 B1
suitable solvent or dispersant, such as water. The particular solvent or dispersant should also be compatible with the
material of the working electrode 22 (e.g., the solvent or dispersant should not dissolve the electrode.)
[0065] One of the most important functions of the sorbent material is to reduce the volume of fluid needed to fill the
sample chamber and corresponding measurement zone of the sensor. The actual volume of sample within the meas-
s urement zone is partially determined by the amount of void space within the sorbent material. Typically, suitable sorbents
consist of about 5% to about 50% void space. Preferably, the sorbent material consists of about 10% to about 25%
void space.
[00661 The displacement of fluid by the sorbent material is advantageous. By addition of a sorbent, less sample is
needed to fill sample chamber 26. This reduces the volume of sample that is required to obtain a measurement and
10 also reduces the time required to electrolyze the sample.
[0067] The sorbent material 34 may include a tab 33 which is made of the same material as the sorbent and which
extends from the sensor, or from an opening in the sensor, so that a sample may be brought into contact with tab 33,
sorbed by the tab, and conveyed into the sample chamber 26 by the wicking action of the sorbent material 34. This
provides a preferred method for directing the sample into the sample chamber 26. For example, the sensor may be
'5 brought into contact with a region of an animal (including human) that has been pierced with a lancet to draw blood.
The blood is brought in contact with tab 33 and drawn into sample chamber 26 by the wicking action of the sorbent
34. The direct transfer of the sample to the sensor is especially important when the sample is very small, such as when
the lancet is used to pierce a portion of the animal that is not heavily supplied with near-surface capillary vessels and
furnishes a blood sample volume of less than 1 ul.
20 [0068] Methods other than the wicking action of a sorbent may be used to transport the sample into the sample
chamber or measurement zone. Examples of such means for transport include the application of pressure on a sample
to push it into the sample chamber, the creation of a vacuum by a pump or other vacuum-producing means in the
sample chamber to pull the sample into the chamber, capillary action due to interracial tension of the sample with the
walls of a thin sample chamber, as well as the wicking action of a sorbent material.
25 [0069] The sensor can also be used in conjunction with a flowing sample stream. In this configuration, the sample
stream is made to flow through a sample chamber. The flow is stopped periodically and the concentration of the analyte
is determined by electrochemical method, such as coulometry. After the measurement, the flow is resumed, thereby
removing the sample from the sensor. Alternatively, sample may flow through the chamber at a very slow rate, such
that all of the analyte is electrolyzed in transit, yielding a current dependent only upon analyte concentration and flow
30 rate.
[0070] Other filler materials may be used to fill the measurement zone and reduce the sample volume. For example,
glass beads can be deposited in the measurement zone to occupy space. Preferably, these filler materials are hy-
drophilic so that the body fluid can easily flow into the measurement zone. In some cases, such as glass beads with
a high surface area, these filler materials may also wick the body fluid into the measurement zone due to their high
35 surface area and hydrophilicity.
[0071] The entire sensor assembly is held firmly together to ensure that the sample remains in contact with the
electrodes and that the sample chamber and measurement zone maintain the same volurne. This is an important
consideration in the coulometric analysis of a sample, where measurement of a defined sample volume is needed.
One method of holding the sensor together is depicted in Figures 1 and 2. Two plates 38 are provided at opposite ends
40 of the sensor. These plates are typically constructed of non-conducting materials such as plastics. The plates are
designed so that they can be held together with the sensor between the two plates. Suitable holding devices include
adhesives, clamps, nuts and bolts, screws, and the like.
Integrated Sample Acquisition and Analyte Measurement Device
45
[0072] In a preferred embodiment of the invention, an analyte measurement device 52 constructed according to the
principles of the present invention includes a sensor 20, as described hereinabove, combined with a sample acquisition
means 50 to provide an integrated sampling and measurement device. The sample acquisition means 50 illustrated
in Figure 6, includes, for example, a skin piercing member 54, such as a lancet, attached to a resilient deflectable strip
so 56 (or other similar device, such as a spring) which may be pushed to inject the lancet into a patient's skin to cause
blood flow.
[0073] The resilient strip 56 is then released and the skin'piercing member 54 retracts. Blood flowing from the area
of skin pierced by member 54 can then be transported, for example^ by the wicking action of sorbent material 34, into
. sensor 20 for analysis of the analyte. The analyte measurement device 52 may then be placed in a reader, not shown,
55 which connects a coulometer or other electrochemicaJ analysis equipment to the electrode tabs 23, 25 to determine
the concentration of the analyte by electroanalytical means.
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Operation of the Sensor
[0074] An electrochemical sensor of the invention is operated in the following manner. A potential is applied across
the working and counter electrodes. The magnitude of the required potential is dependent on the redox mediator The
potential at an electrode where the analyte is electrolyzed is typically large enough to drive the electrochemical reaction
to or near completion, but the magnitude of the potential is, preferably, not large enough to induce significant electro-
chemical reaction of interferents, such as urate, ascorbate, and acetaminophen, that may affect the current measure-
ments. Typically the potential is between about -150 mV and about +400 mV versus the standard calomel electrode
(SCE). Preferably, the potential of the redox mediator is between about -100 mV and +100 mV and, more preferably
the potential is between about -50 mV and +50 mV.
[0075] The potential may be applied either before or after the sample has been placed in the sample chamber. The
potential is preferably applied after the sample has come to rest in the sample chamber to prevent electrolysis of sample
passing through the measurement zone as the sample chamber is filling. When the potential is applied and the sample
is in the measurement zone, an electrical current will flow between the working electrode and the counter electrode
The current is a result of the electrolysis of the analyte in the sample. This electrochemical reaction occurs via the
redox mediator and the optional second electron transfer agent. For many biomolecules, B, the process is described
by the following reaction equations:
nA(ox) + B en2yme > nA(ted). + C
(1)
nA(red) -> nA(ox) + ne" (2)
Biochemical B is oxidized to C by redox mediator species A in the presence of an appropriate enzyme. Then the redox
mediator A is oxidized at the electrode. Electrons are collected by the electrode and the resulting current is measured.
[0076] As an example, one sensor of the present invention is based on the reaction of a glucose molecule with two
non-leachable ferricyanide anions in the presence of glucose oxidase to produce two non-leachable ferrocyanide an-
ions, two protons and gluconolactone. The amount of glucose present is assayed by electrooxidizing the non-leachable
ferrocyanide anions to non-leachable ferricyanide anions and measuring the total charge passed.
[0077] Those skilled in the art will recognize that there are many different reaction mechanisms that will achieve the
same result; namely the electrolysis of an analyte through a reaction pathway incorporating a redox mediator. Equations
(1) and (2) are a non-limiting example of such a reaction.
[0078] In a preferred embodiment of the invention, coulometry is used to determine the concentration of the analyte.
This measurement technique utilizes current measurements obtained at intervals over the course of the assay, to
determine analyte concentration. These current measurements are integrated overtime to obtain the amount of charge,
Q, passed to or from the electrode. Q is then used to calculate the concentration of the analyte by the following equation:
[analyte] = Q/nFV (3)
where n is the number of electron equivalents required to electrolyzethe analyte, F is Faraday's constant (approximately
96,500 coulombs per equivalent), and V is the volume of sample in the measurement zone.
[0079] In one embodiment of the invention, the analyte is completely or nearly completely electrolyzed. The charge
is then calculated from current measurements made during the electrochemical reaction and the concentration of the
analyte is determined using equation (3). The completion of the electrochemical reaction is typically signaled when the
current reaches a steady-state value. This indicates that all or nearly all of the analyte has been electrolyzed. For this
type of measurement, at least 90% of the analyte is typically electrolyzed, preferably, at least 95% of the analyte is
electrolyzed and, more preferably, at least 99% of the analyte is electrolyzed.
[0080] For this method it is desirable that the analyte be electrolyzed quickly. The speed of the electrochemical
reaction depends on several factors, including the potential that is applied between the electrodes and the kinetics of
reactions (1) and (2). (Other significant factors include the size of the measurement zone and the presence of sorbent
In the measurement zone.) In general, the larger the potential, the larger the current through the cell (up to a transport
limited maximum) and therefore, the faster the reaction will typically occur. However, if the potential is too large, other
electrochemical reactions may introduce significant error in the measurement. Typically, the potential between the
10
EP 0 958 495 B1
electrodes as well as the specific redox mediator and optional second electron transfer agent are chosen so that the
analyte will be almost completely electrolyzed in less than 5 minutes, based on the expected concentration of the
analyte in the sample. Preferably, the analyte will be almost completely electrolyzed within about 2 minutes and, more
preferably, within about 1 minute.
5 [0081] In another embodiment of the invention, the analyte is only partially electrolyzed. The current is measured
during the partial reaction and then extrapolated using mathematical techniques known to those skilled in the art to
determine the current curve for the complete or nearly complete electrolysis of the analyte. Integration of this curve
yields the amount of charge that would be passed if the analyte were completely or nearly completely electrolyzed
and, using equation (3), the concentration of the analyte is calculated.
io [0082] The above described methods are based on coulometric analyses, due to the advantages of coulometric
measurements, as described hereinbelow. However, those skilled in the art will recognize that a sensor of the invention
may also utilize potentiometric, amperometnc, voltammetric, and other electrochemical techniques to determine the
concentration of an analyte in a sample. There are, however, disadvantages to using some of these techniques. The
measurements obtained by these non-coulometric methods are not temperature independent as the current and po-
15 tential obtained by the electrolysis of an analyte on an electrode is very sensitive to sample temperature. This presents
a problem for the calibration of a sensor which will be used to measure bioanalytes and other samples at unknown or
variable temperatures.
[0083] In addition, the measurements obtained by these non-coulometric electrochemical techniques are sensitive
to the amount of enzyme provided in the sensor. If the enzyme deactivates or decays over time, the resulting meas-
20 urements will be affected. This will limit the shelf life of such sensors unless the enzyme is very stable.
[0084] Finally, the measurements obtained by non-coulometric electrochemical techniques such as amperometry
will be negatively affected if a substantial portion of the analyte is electrolyzed during the measurement period. An
accurate steady-state measurement can not be obtained unless there Is sufficient analyte so that only a relatively small
portion of the analyte is electrolyzed during the measurement process.
25 [0085] The electrochemical technique of coulometry overcomes these problems. Coulometry is a method for deter-
mining the amount of charge passed or projected to pass during complete or nearly complete electrolysis of the analyte.
One coulometric technique involves electrolyzing the analyte on a working electrode and measuring the resulting cur-
rent between the working electrode and a counter electrode at two or more times during the electrolysis. The electrolysis
is complete when the current reaches a steady state. The charge used to electrolyze the sample is then calculated by
30 integrating the measured currents over time. Because the charge is directly related to the amount of analyte in the
sample there is no temperature dependence of the measurement. In addition, the activity of the redox mediator does
not affect the value of the measurement, but only the time required to obtain the measurement (i.e., less active redox
mediator requires a longer time to achieve complete electrolysis of the sample) so that decay of the mediator over time
will not render the analyte concentration determination inaccurate. And finally, the depletion of the analyte in the sample
35 by electrolysis is not a source of error, but rather the objective of the technique. (However, the analyte need not be
completely electrolyzed if the electrolysis curve is extrapolated from the partial electrolysis curve based on well-known
electrochemical principles.)
[0086] For coulometry to be an effective measurement technique for determining the concentration of an analyte in
a sample, it is necessary to accurately determine the volume of the measured sample. Unfortunately, the volume of
*o the sample in the measurement zone of a small volume sensor (i.e., less than one microliter) may be difficult to accu-
rately determine because the manufacturing tolerances of one or more dimensions of the measurement zone may
have significant variances.
Air-oxldlzable Redox Mediators
45
[0087] Another source of error in a coulometric sensor is the presence of electrochemical reactions other than those
associated with the analyte. In a sensor having a redox mediator, a potential source of measurement error Is the
presence of redox mediator in an unknown mixed oxidation state (i.e., mediator not reproducibly in a known oxidation
state). Redox mediator will then be electrolyzed at the electrode, not in response to the presence of an analyte, but
50 simply due to its initial oxidation state. Referring to equations (1) and (2), current not attributable to the oxidation of
biochemical B will flow due to oxidation of a portion of a redox mediator, A, that is in its reduced form prior to the addition
of the sample. Thus, it is important to know the oxidation state of the analyte prior to introduction of the sample into
the sensor. Furthermore, it is desirable that all or nearly all of the redox mediator be in a single oxidation state prior to
the introduction of the sample into the sensor. ■
55 [0088] Each redox mediator has a reduced form or state and an oxidized form or state. In one aspect of the invention,
it is preferred that the amount of redox mediator in the reduced form prior to the introduction of sample be significantly
smaller than the expected amount of analyte in a sample in order to avoid a significant background contribution to the
measured current. In this embodiment of the invention, the molar amount of redox mediator in the reduced form prior
11
EP 0 958 495 B1
to the introduction of the anaiyte is preferably less than, on a stoichiometric basis, about 10%, and more preferably
less than about 5%, and most preferably less than 1%, of the molar amount of anaiyte for expected anaiyte concen-
trations. (The molar amounts of anaiyte and redox mediator should be compared based on the stoichiometry of the
applicable redox reaction so that if two moles of redox mediator are needed to electrolyze one mole of anaiyte, then
the molar amount of redox mediator in the reduced form prior to introduction of the anaiyte is preferably less than 20%
and more preferably less than about 10% and most preferably less than about 2% of the molar amount of anaiyte for
expected anaiyte concentrations.) Methods for controlling the amount of reduced mediator are discussed below.
[0089] In another aspect of the invention, it is preferred that the relative ratio of oxidized redox mediator to reduced
redox mediator prior to introduction of the sample in the sensor be relatively constant between similarly constructed
sensors. The degree of variation in this ratio between similarly constructed sensors will negatively affect the use of a
calibration curve to account for the reduced mediator, as significant variations between sensors will make the calibration
less reliable. For this aspect of the invention, the percentage of the redox mediator in the reduced form prior to intro-
duction of the sample in the sensor varies by less than about 20% and preferably less than about 1 0% between similarly
constructed sensors.
[0090] One method of controlling the amount of reduced redox mediator prior to the introduction of the sample in the
sensor is to provide an oxidizer to oxidize the reduced form of the mediator. One of the most convenient oxidizers is
0 2 . Oxygen is usually readily available to perform this oxidizing function. Oxygen can be supplied by exposing the
sensor to air. In addition, most polymers and fluids absorb 0 2 from the air unless special precautions are taken. Typically,
at least 90% of an air-oxidizable (i.e., 0 2 oxidizable) mediator is in the oxidized state upon storage or exposure to air
for a useful period of time, e.g., one month or less, and preferably, one week or less, and, more preferably, one day or
less.
[0091 ] Suitable mediators which are both air-oxidizable (i.e., 0 2 -oxidizable) and have electron transfer capabilities
have been described hereinabove. One particular family of useful mediators are osmium complexes which are coor-
dinated or bound to iigands with one or more nitrogen-containing heterocycles. In particular, osmium complexed with
mono-, di- t and polyalkoxy-2,2'-bipyridlne or mono-, di-, and poiyalkoxy-1 , 1 0-phenanthroline, where the alkoxy groups
have a carbon to oxygen ratio sufficient to retain solubility in water, are air-oxidizable. These osmium complexes typ-
ically have two substituted bipyridine or substituted phenanthroline iigands, the two Iigands not necessarily being iden-
tical. These osmium complexes are further complexed with a polymeric ligand with one or more nitrogen-containing
heterocycles, such as pyridine and imidazole. Preferred polymeric Iigands include poly(4-vinyf pyridine) and, more
preferably, poly(1-vinyl imidazole) or copolymers thereof. Os^'-dimethoxy^'-bipyridinekCr^ 2 complexed with a
poly(1 -vinyl imidazole) or poly(4-vinyl pyridine) has been shown to be particularly useful as the Os +2 cation is oxidizable
by 0 2 to Os +3 . Similar results are expected for complexes of Os[4,7-dimethoxy-1 ,1 0-phenanthroIine] 2 CK +2 , and other
mono-, di-, and polyalkoxy bipyridines and phenanthrolines, with the same polymers.
[0092] A complication associated with air-oxidizable mediators arises if the air oxidation of the redox mediator is so
fast that a substantial portion of the analyte-reduced redox mediator is oxidized by 0 2 during an anaiyte assay. This
will result in an inaccurate assay as the amount of anaiyte will be underestimated because the mediator will be oxidized
by the oxidizer rather than by electrooxidation at the electrode. Thus, it is preferred that the reaction of the redox
mediator with 0 2 proceeds more slowly than the electrooxidation of the mediator. Typically, less than 5%, and preferably
less than 1%, of the reduced mediator should be oxidized by the oxidizer during an assay.
[0093] The reaction rate of the air oxidation of the mediator can be controlled through choice of an appropriate
complexing polymer. For example, the oxidation reaction is much faster for Os[4,4 , -dimethoxy-2,2 , -bipyridine] 2 CI 4/+2
coordinatively coupled to poly(1 -vinyl imidazole) than for the same Os complex coupled to pofy(4-vlnyl pyridine). The
choice of an appropriate polymer will depend on the expected anaiyte concentration and the potential applied between
the electrodes, both of which determine the rate of the electrochemical reaction.
[0094] Thus, in one embodiment of the invention, the preferred redox mediator has the following characteristics: 1)
the mediator does not react with any molecules in the sample or in the sensor other than the anaiyte (optionally, via a
second electron transfer agent); 2) nearly all of the redox mediator is oxidized by an oxidizer such as 0 2 prior to
introduction of the sample in the sensor; and 3) the oxidation of the redox mediator by the oxidizer is slow compared
to the electrooxidation of the mediator by the electrode.
[0095] Alternatively, if the redox mediator is to be oxidized in the presence of the anaiyte and electroreduced at the
electrode, a reducer rather than an oxidizer would be required. The same considerations for the appropriate choice of
reducer and mediator apply as described hereinabove for the oxidizer.
[0096] The use of stable air-oxidizable redox mediators in the electrochemical sensors of the invention provides an
additional advantage during storage and packaging. Sensors of the invention which include air oxidizable redox me-
diators can be packaged in an atmosphere containing molecular oxygen and stored for long periods of time, e.g.,
greater than one month, while maintaining more than 80% and preferably more than 90% of the redox species in the
oxidized state.
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EP 0 958 495 B1
Optical Sensors
[0097J The air-oxidizable redox species of the present invention can be used in other types of sensors. The osmium
complexes described hereinabove are suitable for use in optical sensors, due to the difference in the absorption spectra
and fluorescence characteristics of the complexed Os +2 and Os* 3 species. Absorption, transmission, reflection, or
fluorescence measurements of the redox species will correlate with the amount of analyte in the sample (after reaction
between an analyte and the redox species, either directly, or via a second electron transfer agent such as an enzyme).
In this configuration, the molar amount of redox mediator should be greater, on a stoichiometric basis, than the molar
amount of analyte reasonably expected to fill the measurement zone of the sensor.
[0098] Standard optical sensors, including light-guiding optical fiber sensors, and measurement techniques can be
adapted for use with the air-oxidizable mediators For example, the optical sensors of the invention may include a light-
transmitting or light reflecting support on which the air-oxidizable redox species, and preferably an analyte-responsive
enzyme, is coated to form a film. The support film forms one boundary for the measurement zone in which the sample
is placed. The other boundaries of the measurement zone are determined by the configuration of the cell. Upon filling
the measurement zone with an analyte-containing sample, reduction of the air-oxidizable mediator by the analyte,
preferably via reaction with the analyte-responsive enzyme, causes a shift in the mediator's oxidation state that is
detected by a change in the light transmission, absorption, or reflection spectra or in the fluorescence of the mediator
at one or more wavelengths of light.
Multiple Electrode Sensors and Calibration
[0099] Multiple electrode sensors may be used for a variety of reasons. For example, multiple electrode sensors
may be used to test a variety of analytes using a single sample. One embodiment of a multiple electrode sensor has
one or more sample chambers which in turn may contain one or more working electrodes 22 with each working electrode
22 defining a different measurement zone. One or more of the working electrodes have the appropriate chemical
reagents, for example, an appropriate enzyme, to test a first analyte and one or more of the remaining working elec-
trodes have appropriate chemical reagents to test a second analyte. For example, a multiple electrode sensor might
include 1) one or more working electrodes having glucose oxidase in the sensing layer to determine glucose concen-
tration and 2) one or more working electrodes having lactate oxidase in the sensing layer to determine lactate concen-
tration. Other combinations are also possible.
[0100] Multiple electrode sensors may also be used to improve the precision of the resulting readings. The meas-
urements from each of the working electrodes (all or which are detecting the same analyte) can be averaged together
to obtain a more precise reading. In some cases, measurements may be rejected if the difference between the value
and the average exceeds a threshold limit. This threshold limit may be, for example, determined based on a statistical
parameter, such as the standard deviation of the averaged measurements. The average may then be recalculated
while omitting the rejected values. Furthermore, subsequent readings from an electrode that produced a rejected value
may be ignored in later tests if it is assumed that the particular electrode is faulty. Alternatively, a particular electrode
may be rejected only after having a predetermined number of readings rejected based on the readings from the other
electrodes.
[0101] In addition to using multiple electrode sensors to increase precision, multiple measurements may be made
at each electrode and averaged together to increase precision. This technique may also be used with a single electrode
sensor to increase precision.
[0102] Errors in assays may occur when mass produced sensor are used because of variations in the volume of the
measurement zone of the sensors. Two of the three dimensions of the measurement zone, the length and the width,
are usually relatively large, between about 1-5 mm. Electrodes of such dimensions can be readily produced with a
variance of 2% or less. The submicroliter measurement zone volume requires, however, that the third dimension be
smaller than the length or width by one or two order of magnitude. As mentioned hereinabove, the thickness of the
sample chamber is typically between about 0.1 and about 0.01 mm. Manufacturing variances in the thickness may be
as large or larger than the desired thickness. Therefore, it is desirable that a method be provided to accommodate for
this uncertainty in the volume of sample within the measurement zone.
[0103] In one embodiment of the invention, depicted in Figure 5, multiple working electrodes 42, 44, 46 are provided
on a base material 48. These electrodes are covered by another base, not shown, which has counter electrodes, not .
shown, disposed upon ft to provide multiple facing electrode pairs. The variance in the separation distance between
the working electrode and the counter electrode among the electrode pairs on a given sensor is significantly reduced,
because the working electrodes and counter electrodes are each provided on a single base with the same spacer 28
between each electrode pair (see Figure 3).
[0104] One example of a multiple electrode sensor that can be used to accurately determine the volume of the
measurement zones of the electrode pairs and also useful in reducing noise is presented herein. In this example, one
13
EP 0 958 495 B1
o, the working electrodes 42 is prepared with a non-leachable ■^^^ESSES S2£25
2 anv SdtaMMn electrode 46 (or any of the other electrodes 42, 44 in the absence of sorbent matenal)
may be used for convenience.
EXAMPLES
roiOSI The invention will be further characterized by the following examples. These examples are nol tmeant tolimit
toZ^SZSZ* which has been fully set forth in the foregoing description. Variations w,th,n the concepts of
the invention are apparent to those skilled in the art.
Example 1
Preparation of a Small Volume In wlro Sensor for the Determination of Glucose Concentration
14
EP 0 958 495 B1
mediator was approximately 1:15. The mediator was deposited on the working electrode in a layer having a thickness
of 0.6 urn and a diameter of 4 mm. The coverage of the mediator on the electrode was about 60 u.g/cm 2 (dry weight).
A spacer material was placed on the electrode surrounding the mediator-covered surface of the electrode. The spacer
was made of poly(tetrafluoroethylene) (PTFE) and had a thickness of about 0.040 mm.
5 [0112] A sorbent material was placed in contact with the mediator-covered surface of the working electrode. The
sorbent was made of nylon (Tetko Nitex nylon 3-10/2) and had a diameter of 5 mm, a thickness of 0.045 mm, and a
void volume of about 20%. The volume of sample in the measurement zone was calculated from the dimensions and
characteristics of the sorbent and the electrode. The measurement zone had a diameter of 4 mm (the diameter of the
mediator covered surface of the electrode) and a thickness of 0.045 mm (thickness of the nylon sorbent) to give a
.10 volume of 0.57 ul. Of this space, about 80% was filled with nylon and the other 20% was void space within the nylon
sorbent. This resulting volume of sample within the measurement zone was about 0.11 u.L.
[0113] A counter/reference electrode was placed in contact with the spacer and the side of the sorbent opposite to
the working electrode so that the two electrodes were facing each other. The counter/reference electrode was con-
structed on a Mylar™ film having a thickness of 0.175 mm and a diameter of about 2.5 cm onto which a 12 micron
15 thick layer of silver/silver chloride having a diameter of about 1 cm was screen printed.
[0114) The electrodes, sorbent, and spacer were pressed together using plates on either side of the electrode as-
sembly. The plates were formed of polycarbonate plastic and were securely clamped to keep the sensor together. The
electrodes were stored in air for 48 hours prior to use.
[01 1 5] Tabs extended from both the working electrode and the counter/reference electrode and provided for an elec-
ta trical contact with the analyzing equipment. A potentiostat was used to apply a potential difference of +200mV between
the working and counter/reference electrodes, with the working electrode being the anode. There was no current flow
between the electrodes in the absence of sample, which was expected, as no conductive path between the electrodes
was present.
[01 16] The sample was introduced via a small tab of nylon sorbent material formed as an extension from the nylon
25 sorbent in the sample chamber. Liquid was wicked into the sorbent when contact was made between the sample and
the sorbent tab. As the sample chamber filled and the sample made contact with the electrodes, current flowed between
the electrodes. When glucose molecules in the sample came in contact with the glucose oxidase on the working elec-
trode, the glucose molecules were electrooxidized to gluconolactone. The osmium redox centers in the redox mediator
then reoxidizedthe glucose oxidase, the osmium centers were in turn reoxidized by reaction with the working electrode.
30 This provided a current which was measured and simultaneously integrated by a coulometer. (EG&G Princeton Applied
Research Model #1 73)
[0117] The electrochemical reaction continued until the current reached a steady state value which indicated that
greater than 95% of the glucose had been electroreduced. The current curve obtained by measurement of the current
at specific intervals was integrated to determine the amount of charge passed during the electrochemical reaction.
35 These charges were then plotted versus the known glucose concentration to produce a calibration curve.
[0118] The sensor was tested using 0.5 \il aliquots of solutions containing known concentrations of glucose in a
buffer of artificial cerebrospinal. fluid or in a control serum (Baxter-Dade, Monitrol Level 1 , Miami, FL) in the range of 3
to 20 mM glucose. The artificial cerebrospinal fluid was prepared as a mixture of the following salts: 126 mM NaCI,
27.5 mM NaHC0 3 , 2.4 mM KCI, 0.5 mM KH 2 P0 4 , 1 .1 mM CaCI 2 .2H 2 0, and 0.5 mM NagSC^.
^0 [01 1 9] The results of the analyses are shown in Table 1 and in Figure 7. In Table 1 , Q avg is the average charge used
to electrolyze the glucose in 3-6 identical test samples (Figure 7 graphs the charge for each of the test samples) and
the 90% rise time corresponds to the amount of time required for 90% of the glucose to be electrolyzed. The data show
a sensor precision of 1 0- 20%, indicating adequate sensitivity of the sensor for low glucose concentrations, as well as
in the physiologically relevant range (30 u,g/dL - 600 ng/dL).
45
SO
55
15
EP 0 958 495 B1
TABLE 1
Sensor Results Using Glucose Oxidase
YV. V" T. >.< i V.irVx! : ; :>; v ; ; : %' • * -
N^be^.^pf i>
• *. ••. -. . ....
.K90% ri^e=tinj6-
buffer only
4
9.9 ± 1.8
13 ± 6
3 mM glucose/buffer
5
17.8± 3.5
19± 5
6 mM glucose/buffer "
4
49.4 ± 4.9
25± 3
10 mM glucose/buffer
6
96.1 ± 12.4
36±17
15 mM glucose/buffer
5
205.2 ±75.7
56±23
20 mM glucose/buffer
4
255.7*41.0
62±17
•sy^r:-'. i"?^'^ ;
4.2 mM glucose/serum
3
44.2 ± 4.3
44± 3
15.8 mM glucose/serum
3
218.2 ±57.5
72 ±21
[0120J The average measured values of glucose concentration were fit by one or more equations to provide a cali-
bration curve. Figure 8 shows the calibration curves for the glucose/buffer data of Table 1 . One of the 15.0 mM glucose
measurements was omitted from these calculations because it was more than two standard deviations away from the
average of the measurements. The higher glucose concentrations (1 0-20 mM) were fit by a linear equation. The lower
glucose concentrations were fit by a second order polynomial.
[0121] Figure 9 shows the data of Table 1 plotted on an error grid developed by Clarke, et al. Diabetes Care, 5,
622-27, 1 987; for the determination of the outcome of errors based on inaccurate glucose concentration determination.
The graph plots "true" glucose concentration vs. measured glucose concentration, where the measured glucose con-
centration is determined by calculating a glucose concentration using the calibration curves of figure 8 for each data
point of figure 7. Points in zone A are accurate, those in zone B are clinically acceptable, and those in zones C, D, and
E lead to increasingly inappropriate and finally dangerous treatments.
[0122] There were 34 data points. Of those data points 91 % fell in zone A, 6% In zone B, and 3 % in zone C. Only
one reading was determined to be in zone C. This reading was off-scale and is not shown in figure 9. Thus, 97% of
the readings fell in the clinically acceptable zones A and B.
[0123] The total number of Os atoms was determined by reducing all of the Os and then electrooxidizing it with a
glucose-free buffer in the sample chamber. This resulted in a charge of 59.6 ± 5.4 u.C. Comparison of this result with
the glucose-free buffer result in Table 1 indicated that less than 20% of the Os is in the reduced form prior to introduction
of the sample. The variability in the quantity of osmium in the reduced state is less than 5% of the total quantity of
osmium present.
Example 2
Response of the Glucose Sensor to Interferents
[0124] A sensor constructed in the same manner as described above for Example 1 was used to determine the
sensor's response to interferents. The primary electrochemical interferents for blood glucose measurements are ascor-
bate, acetaminophen, and urate. The normal physiological or therapeutic (in the case of acetaminophen) concentration
ranges of these common interferents are:
ascorbate: 0.034 - 0.114 mM
16
EP 0 958 495 B1
acetaminophen: 0.066 - 0.200 mM
urate (adult male): 0.27 - 0.47 mM
Tietz, in: Textbook of Clinical Chemistry, C.A. Burtis and E.R. Ashwood, eds., W.B. Saunders Co., Philadelphia 1994,
5 pp. 2210-12.
[0125] Buffered glucose-free interferent solutions were tested with concentrations of the interferents at the high end
of the physiological or therapeutic ranges listed above. The injected sample volume in each case was 0.5 u.L. A potential
of +1 00 m V or +200 mV was applied between the electrodes. The average charge (Q avg ) was calculated by subtracting
an average background current obtained from a buffer-only (i.e., interferent-free) solution from an average signal re-
10 corded with interferents present. The resulting average charge was compared with the signals from Table 1 for 4 mM
and 1 0 mM glucose concentrations to determine the percent error that would result from the interferent.
TABLE 2
Interferent Response of Glucose Sensors
/E; : (mV);,
■:: : ^.::t
"mM.'glucpse'
Error.@:lX!; .
mM glucose
0.114mM ascorbate
100
4
0.4
2%
<1%
0.1 14 mM ascorbate
200
4
-0.5
2% •
<1%
0.2 mM acetaminophen
100
4
0.1
<1%
<l%
0.2 mM acetaminophen
200
4
1.0
5%
1%
0.47 mM urate
100
4
6.0
30%
7%
0.47 mM urate
200
4
18.0
90%
21%
[01 26] These results indicated that ascorbate and acetaminophen were not significant interferents for the glucose
sensor configuration, especially for low potential measurements. However, urate provided significant interference. This
interference can be minimized by calibrating the sensor response to a urate concentration of 0.37 mM, e.g., by sub-
4 $ tracting an appropriate amount of charge as determined by extrapolation from these results from all glucose measure-
ments of the sensor. The resulting error due to a 0.10 mM variation in urate concentration (the range of urate concen-
tration is 0.27 - 0.47 in an adult male) would be about 6% at 4 mM glucose and 1 0OmV.
Example 3
50
Sensor with Glucose Dehydrogenase
[01 27] A sensor similar to that described for Example 1 was prepared and used for this example, except that glucose
oxidase was replaced by pyrroloquinoline quinone glucose dehydrogenase and a potential of only +1 00 mV was applied
55 as opposed to the +200 mV potential in Example 1 . The results are presented in Table 3 below and graphed in Fig. 1 0.
17
EP 0 958 495 B1
TABLE 3
Sensor Results Using Glucose Dehydrogenase
"iftSv
buffer
4
21.7 ± 5.2
14±3
3 mM glucose/buffer
4
96.9 ±15.0
24±6
6 mM glucose/buffer
4
190.6 ±18.4
26±6
1 0 mM glucose/buffer
4
327.8 ±69.3
42±9
[0128J The results indicated that the charge obtained from the glucose dehydrogenase sensor was much larger than
for the comparable glucose oxidase sensor, especially for low concentrations of glucose. For 4 mM glucose concen-
trations the measurements obtained by the two sensors differed by a factor of five. In addition, the glucose dehydro-
genase sensor operated at a lower potential, thereby reducing the effects of interferent reactions.
[01291 In addition, the results from Table 3 were ail fit by a linear calibration curve as opposed to the results in
Example 1, as shown in Fig. 10. A single linear calibration curve is greatly preferred to simplify sensor construction
and operation.
[0130] Also, assuming that the interferent results from Table 2 are applicable for this sensor, all of the interferents
would introduce an error of less than 7% for a 3 mM glucose solution at a potential of 100 mV.
Example 4
Determination of Lactate Concentration in a Fluid Stream
[0131] The sensor of this Example was constructed using a flow cell (BioAnalytical Systems, Inc. # MF-1 025) with
a glassy carbon electrode. A redox mediator was coated on the electrode of the flow cell to provide a working electrode.
In this case, the redox mediator was a polymer formed by complexing poly(1 -vinyl imidazole) with Os(4,4'-dimethyl-
2,2 , -bipyridine) 2 CI 2 with a ratio of 1 osmium for every 15 imidazole functionalities. Lactate oxidase was cross-linked
with the polymer via polyethylene glycol diglycidyl ether. The mediator was coated onto the electrode with a coverage
of 500 u.g/cm 2 and a thickness of 5 u.m. The mediator was covered by a polycarbonate track-etched membrane (Os-
monics-Poretics #10550) to improve adherence in the flow stream. The membrane was then overlaid by a single 50
urn thick spacer gasket (BioAnalytical Systems, Inc. #MF-1062) containing a void which defined the sample chamber
and corresponding measurement zone. Assembly of the sensor was completed by attachment of a cell block (BioAn-
alytical Systems, Inc. #MF-1005) containing the reference and auxiliary electrodes of the flow cell.
[01 32] The sample chamber in this case corresponded to a 50 ujti thick cylinder (the thickness of the spacer gasket)
in contact with a mediator-coated electrode having a surface area of 0.031 cm 2 . The calculated volume of sample in
the measurement zone of this sensor was approximately 0. 1 6 u,L.
[0133] The flow rate of the fluid stream was 5 nL/min. A standard three electrode potentiostat was attached to the
cell leads and a potential of +200 mV was applied between the redox mediator-coated glassy carbon electrode and
the reference electrode. This potential was sufficient to drive the enzyme-mediated oxidation of lactate.
[01 34] As the fluid stream flowed through the sensor, a steady-state current proportional to the lactate concentration
was measured. At periodic intervals the fluid flow was stopped and current was allowed to flow between the electrodes
until approximately ail of the lactate in the measurement zone was electrooxidized, as indicated by the achievement
of a stabilized, steady-state current. The total charge, Q, required for lactate electrooxidation was found by integration
of the differential current registered from the flow stoppage until the current reached a steady-state. The concentration
was then calculated by the following equation:
[lactate] » Q/2FV (4)
where V is the volume of sample within the measurement zone and F is Faraday's constant.
18
EP 0 958 495 B1
[01 35] This assay was performed using lactate solutions having nominal lactate concentrations of t .0, 5.0, and 1 0.0
mM. The measured concentrations for the assay were 1 .9, 5.4, and 8.9 mM respectively.
Example 5
Determination of the Oxidation State of Os(4,4 , -dimethoxy-2,2'-bIpyrldlne) 2 CK*2 Complexed with poly(1-vlnyl
imidazole)
[0136] A sensor having a three electrode design was commercially obtained from Ecossensors Ltd., Long Hanbor-
ough, England, under the model name "large area disposable electrode". The sensor contained parallel and coplanar
working, reference and counter electrodes. The working surface area (0.2 cm 2 ) and counter electrodes were formed
of printed carbon and the reference electrode was formed of printed Ag/AgCI. A redox mediator was coated on the
carbon working electrode. The redox mediator was formed by complexation of poly(1 -vinyl imidazole) with Os(4,4 l -
dimethoxy-2,2 , -bipyridine) 2 CI 2 in a ratio of 15 imidazole groups per Os cation followed by cross linking the osmium
polymer with glucose oxidase using polyethylene glycol diglycidyl ether.
[01 37] The electrode was cured at room temperature for 24 hours. The coplanar electrode array was then immersed
in a buffered electrolyte solution, and a potential of +200 mV (sufficient for conversion of Os(ll) to Os(lll),) was applied
between the working electrode and the reference electrode.
[01 38] Upon application of the potential, an undetectable charge of less than 1 u€ was passed. Subsequent reduction
and reoxidation of the redox mediator yielded a charge for conversion of all Os from Os(ll) to Os(lll) of 65 u.C. Therefore,
more than 98% of the Os cations in the redox mediator were in the desired oxidized Os(lll) state.
Example 6
Determination of the Oxidation State of the Os(4 l 4 , -dimethoxy.2,2 , -bipyridlne) 2 CI^ +2 Complexed with poly
(4-vinyl pyridine)
[0139] A similar experiment to that of Example 5 was conducted with the same working/counter/reference electrode
configuration except that the redox mediator on the working electrode was changed to a complex of Os(4,4'-dimethoxy-
2,2 , -blpyridine) 2 CI 2 with poly(4-vinyl pyridine), with 12 pyridine groups per Os cation, cross linked with glucose oxidase
via polyethylene glycol diglycidyl ether.
[0140] Two sensors were constructed. The electrodes of the two sensors were cured at room temperature for 24
hours. The electrodes were then immersed in a buffered electrolyte solution and a potential of +200 mV was applied
between the working and reference electrodes.
[0141] Upon application of the potential to the electrodes, a charge of 2.5 u,C and 3,8 \iO was passed in the two
sensors, respectively. Subsequent reduction and reoxidation of the redox mediators yielded oxidation charges of 27.9
u.C and 28.0 uXl, respectively. Therefore, the sensors originally contained 91% and 86% of the Os cations in the de-
sirable oxidized Os(lll) state.
Example 7
Optical Sensor
[0142] An optical sensor is constructed by applying a film of redox polymer with crosslinked enzyme onto a light-
transparent support such as a glass slide. The quantity of redox mediator is equal to or greater than (in a stoichiometric
sense) the maximum quantity of anafyte expected to fill the measurement zone! The spacer material, sorbent and
facing support are securely clamped. The sample chamber is adapted to transmit light through the assembled sensor
to an optical density detector or to a fluorescence detector. As sample fills the sample chamber and the redox mediator
is oxidized, changes in the absorption, transmission, reflection or fluorescence of the redox mediator in the chamber
are correlated to the amount of glucose in the sample.
Example 8
Blood Volumes from Upper Arm Lancet Sticks
[0143] The forearm of a single individual was pierced with a lancet multiple times in order to determine the repro-
ducibility of bipod volumes obtained by this method. Despite more than thirty lancet sticks in the anterior portion of
each forearm and the dorsal region of the left forearm, the individual identified each stick as virtually painless.
19
EP 0 958 495 B1
[01 44] The forearm was pierced with a Payless Color Lancet. The blood from each stick was collected using a 1 uL
capillary tube, and the volume was determined by measuring the length of the blood column. The volumes obtained
from each stick are shown below in Table 4,
Table 4
Volume of Lancet Sticks
Left Anterior Forearm, (nL)
Right Anterior Forearm, (nL)
Left Dorsal Forearm, (nL)
1
180
190
180
2
250
180
300
3
170
120
310
4
150
100
300
5
100
210
60
6
50
140
380
. 7
90
120
220
8
130
140
200
9
120
100
380
10
100
320
11
260
.12
250
13
280
.14
260
Avg.
138±58nL
140 + 40 nL
264 ±83 nL j
[0145] The invention has been described with reference to various specific and preferred embodiments and tech-
mques. However, it will be apparent to one of ordinarily skill in the art that many variations and modifications may be
made while remaining within the scope of the invention.
Claims
An electrochemical sensor for determining the concentration of an analyte in a sample, the sensor comprising;
at least one working electrode (22) ;
at least one counter electrode (24) ;
which electrodes, in use, are connected to external electronics of a measuring device;
at least one sample chamber (26), wherein the at least one sample chamber is
(i) a sample chamber for holding the sampie in electrolytic contact with the working electrode and sized
to contain no more than about 1uJ of sample; or
(ii) a sample chamber comprising at least one measurement zone sized to contain no more than about
1 u.l of sample, wherein the measurement zone is bounded on at least two sides by the working and counter
electrodes;
said sensor containing a non-leachable redox mediator on the working electrode;
and
said sensor optionally containing a sorbent material (34) disposed at least partially within the sample chamber
or at least partially within the measurement zone.
The sensor of claim 1, wherein the working electrode and the counter electrode comprise metal or carbon and
the sensor includes the non-leachable redox mediator on the working electrode.
20
EP 0 958 495 B1
3. The sensor of claim 2, wherein the sensor further comprises a second electron transfer agent, preferably an enzyme
such as glucose oxidase, on the working electrode, wherein the second transfer agent is optionally immobilized
on the working electrode and/or is optionally non-leachable.
4. The sensor of daim 1 , wherein the working electrode and the counter electrode comprise metal or carbon, and
the sensor includes the sorbent material disposed at least partially within the sample chamber or at least partially
within the measurement zone.
5. The sensor of any of claims I through 4, wherein the sample chamber or, the measurement zone, is sized to contain
no more than about 0.5 uX, preferably no more than about 0.2 pi, preferably no more than about 0. 1 u.L of sample.
6. The sensor of any of claims 1 through 5, wherein the redox mediator is immobilized on the working electrode.
7. The sensor of any of ciaims 1 through 6, wherein the redox mediator is an air-oxidizable redox mediator, preferably
with at least 90% of the air-oxidizable redox mediator in an oxidized state prior to introduction of sample.
8. The sensor of any of claims 1 through 7, wherein the redox mediator comprises a transition metal complex, such
as an osmium, ruthenium, iron, or cobalt complex; and preferably is osmium complexed with at least one ligand
having a nitrogen-containing heterocycle, such as 2,2 , -bipyridine, 4 ) 4 , -dimethyl-2 J 2 , -bipyridine, 4,4 , -dialkoxy-2,2 , -
bipyridine, 1,10-phenanthroline, 4,7-dimethyl-1,10-phenanthroline, 4,7-dialkoxy-1 ,1 0-phenanthroline, or deriva-
tives thereof.
9. The sensor of claim 8, wherein the ligand having a nitrogen -containing heterocycle comprises 2,2 , -bipyridine,
1 ,1 0-phenanthroline, or a derivative thereof; such as a mono-, di-, or polyalkoxy derivative of 2,2'-bipyridine or
1 ,1 0-phenanthroline, wherein the carbon to oxygen ratio of the alkoxy functional groups is sufficient to retain sol-
ubility of the transition metal complex in water prior to crossiinking.
10. The sensor of claim 8 or 9, wherein the carbon to oxygen ratio of the alkoxy functional groups is sufficient to retain
solubility of the transition metal complex in water prior to crossiinking.
11. The sensor of any of claims I through 10, wherein the sorbent material has a void volume of about 5% to 50%,
preferably about 1 0% to 25%, of the total volume of the sorbent material.
12. The sensor of any of ciaims 1 through 11 , wherein the sensor further comprises one or more additional working
electrodes.
13. The sensor of any of claims 1 through 12, wherein at least one working electrode and at least one counter electrode
form at least one facing electrode pair, optionally wherein each pair is responsive to a specific analyte.
14. The sensor of claim 13, wherein the working electrode and counter electrode have a separation distance of no
more than about 0.2 mm, or no more than about 0.1 mm, or no more than about 0.05 mm.
15. The sensor of claim 13, wherein at least one facing electrode pair comprises a working electrode including non-
leachable enzyme and redox mediator, and wherein at least one other facing electrode pair comprises a working
electrode including non-leachable redox mediator in the absence of the enzyme.
16. The sensor of claim 1 5, further comprising a third electrode (46) pair having no redox mediator or enzyme on the
working electrode.
17. The sensor of any of claims 1 through 16, wherein the working electrode comprises a first arm (122) and the
counter electrode comprises a second arm (124), a portion of the first arm overlapping a portion of the second
arm, wherein the measurement zone or, the sample chamber* comprises a region (21) between the overlapping
portions of the first and second arms.
18. The sensor of claim 1 7, wherein the first arm has an extra length and the overlapping portion of the first arm has
a width, wherein a ratio of the extra length of the first arm to the width of the overlapping portion of the first arm
ranges from 0.1:1 to 50:1, preferably from 1 :1 to 15:1, and more preferably from 4:1 to 10:1.
21
EP 0 958 495 B1
19. The sensor of claim 1 8, wherein the first and second arms intersect at an angle greater than 0 degrees.
20 ' ^X^dT 8 1 ,hr ° U9h 1 9 ' Whercin materia ' C ° mpriSeS 8 powdered — Opposed
^m^dt'.S t Where 'r r m ^ ,aC ' n9 Paif COmpHseS a base materiaI (102) having a
recess (104) and at least one of the working electrode and the counter electrode disposed In the recess.
22 wh!rt!nT? l any ° ,ClaimS 1 thr ° U9h 21 - furthe r comprising afiller material, preferably a hydrophilic filler material
J!?! : f " ,er material iS d l Sp ° Sed in the cement «"e or in the sample chaLJ to dec ease a voZe
of the measurement zone or the sample chamber available for the sample. urease a volume
23. The sensor of claims 1 through 22, wherein the sensor produces a signal in a buffer solution contains 10 mM
giucose that is at least 9 times greater than a signal produced in a buffer solution w^h no glucose '
24 ' l^°K d f °- d f tem,ining a concentration of an analyte in a sample, comprising contacting the sample with the
°' ^ ° f C ' aimS 1 thr ° U9h * "* - — tion 9 of toTS&I the 6
25 " mir.?° d 0 t?! aim 24 \ wherein the step is performed by contacting the sample with the sorbent
matenal to w.ck the sample into the sample chamber or the measurement zone.
26. The method of claim 24, wherein the sample chamber fills by capillary action.
27. The method of claim 24, wherein the step of determining the concentration of the anafyte comprises:
electrolyzing at least 90% of analyte present in the measurement zone by applying a potential across tha
ssss^ elec,rode8, preferably ,n ,ess than about 5 minutes -
determining an electrical charge used to electrolyze the analyte- and
correlating the electrical charge with the concentration of the analyte In the sample-
wherein the determining step is optionally performed by the following steps:
measuring a current generated at the working electrode at two or more times as the analyte is electrolyzed;
integrating the measured currents overtime to obtain the electrical charge used to electrolyze the analyte.
28 7heTeSo^
Itlt? 3 P ° rti0n °' analyte by apP * in9 3 P0ten,ial across the workin 9 and the counter electrode-
measunng a current generated at the working electrode at two or more times during the electrolys?
extrapolating a current curve based on the measured currents- cuoiysis,
thelntjte! ana Urrem °™ "™ * ^ ^ t0 e,ectro ^ e at least 9 °% °'
correlating the electrical charge with the concentration of the analyte in the sample.
30. The method of claim 29, wherein the step of determining the concentration ol analyte comprises the steps of:
^Zh" 9 substant,al| ysimultaneously, and at two ormoretimes.afirst current generated at thefirst electrode
pair and a second current generated at the second electrode pair; eiectroae
integrating the measured first currents over time to obtain a first charge-
integrating the measured second currents over time to obtain a second charge-
22
EP 0 958 495 B1
subtracting the second charge from the first charge to obtain a noise-reduced charge; and
correlating the concentration of the analyte to the noise-reduced charge.
31. The method of claim 24, the method comprising, prior to the contacting and determining steps, the further steps of:
providing the electrochemical sensor of any of claims 1 through 23, wherein the sensor has one or more facing
electrode pairs, each electrode pair comprising a working electrode, a counter electrode, and a measurement
zone between the working electrode and the counter electrode;
measuring a capacitance of at least one of the electrode pairs; and
calculating the volume of the measurement zone from the capacitance measurement.
32. The method of claim 24, wherein the sensor includes a redox mediator on the working electrode, and the molar
amount of redox mediator in a reduced form prior to introduction of the sample is less than, on a stoichiometric
basis, 5% of the expected molar amount of the analyte to be electrolyzed; and wherein the step of determining
the concentration of analyte comprises
electrolyzing less than about 1 \lL of sample.
33. The method of claim 32, wherein the sensor comprises at least two working electrodes, each working electrode
having an associated measurement zone, and wherein the step of determining the concentration of the analyte
comprises averaging measurements from the at least two working electrodes, and optionally further comprises
eliminating measurements which exceed a threshold value, and optionally further comprises reaveraging the meas-
urements without the eliminated measurements.
34. An analyte measurement system comprising:
sample acquisition means, preferably a skin-piercing member (54), such as a lancet, for producing a patient
sample;
the electrochemical sensor of any one of claims 1 through 23; and
optionally a transport means, such as a wicking material, a capillary chamber, a sorbent material or a pump,
for transporting the sample produced by the sample acquisition means to the sample chamber or the meas-
urement zone.
35. The system of claim 34 wherein the skin piercing member is integral with the sensor.
36. A method for measuring analyte in a patient sample, the method comprising:
contacting the patient with an analyte measuring system, the system comprising:
sample acquisition means for producing a patient sample; and
the electrochemical sensor of any of claims 1 through 23, for measuring analyte in the sample;
acquiring a sample using the sample acquisition means;
transporting a portion of the sample to the measurement zone or sample chamber of the electrochemical
sensor, wherein the transporting step optionally comprising wicking the sample into the measurement zone
or sampler chamber using the sorbent material; and
determining the concentration of the analyte in the sample by oculometry.
37. The method of claim 36, which the sample acquisition means is integral with the sensor.
38. The method of claims 36 or 37, wherein the sample acquisition means comprises a skin-piercing member, such
as a lancet, and the step of acquiring a sample comprises piercing the patient's skin at a location other than at a
finger of the patient to produce a sample.
39. A method of determining a concentration of an analyte in a sample, the method comprising steps of:
connecting the electrodes of an electrochemical sensor comprising a working electrode, a counter electrode,
and a sample chamber for holding the sample in electrolytic contact with the working electrode and counter
electrode, to external electronics of a measuring device;
23
EP0 958 495 B1
contacting the sample with the sensor;
holding the sample within the sample chamber in a non-flowing manner;
applying a potential between the working and counter electrodes to electrolyze the anafyte in a portion of the
sample within the sample chamber, wherein the portion of the sample in which the analyte is electrolyzed has
5 a volume of less than 1
measuring current generated by the electrochemical sensor at a plurality of times while the analyte in the
portion of the sample is being electrolyzed; and
determining, by coulometry, a concentration of the analyte in the sample using the measured currents.
10 40. The sensor of claim 7, wherein the sensor further comprises a second electron transfer agent, such as an enzyme,
coated on the support and in contact with the redox mediator, wherein the second electron transfer agent is op-
tionally immobilized on the support.
41. A method for determining a concentration of an analyte in a sample, comprising the steps of:
15
contacting the sample with the sensor of claim 7 or claim 40; and
correlating the concentration of the analyte in the sample to a change in oxidation state of the redox mediator
in the presence of analyte.
20 42. The method of claim 41 , wherein the sensor comprises an optical sensor, and the step of correlating the concen-
tration of the analyte comprises:
irradiating the redox mediator with light;
measuring the response of the redox mediator to irradiation by light; and
25 correlating the concentration of the analyte to the measured response of the redox mediator.
43. The method of claim 42, wherein the step of measuring the response of the redox mediator to irradiation by light
comprises measuring the absorption, or the transmittance, of the irradiated light by the redox mediator.
30 44. The method of claim 42, wherein the step of measuring the response of the redox mediator to irradiation by light
comprises measuring the fluorescence of the redox mediator after irradiation by light, or measuring the reflection
of light by the redox mediator.
45. The method of claim 41 , wherein the sensor comprising an electrochemical sensor, and the step of correlating the
35 concentration of the analyte comprises:
applying an electrical potential across the redox mediator;
measuring a current at one or more intervals, the current being generated in response to the electrolysis of
the redox mediator in the presence of the analyte; and
40 correlating the concentration of the analyte to the measured current.
46. The method of claim 41 , wherein the redox mediator comprises a transition metal complex, such as an osmium
complex, and preferably, osmium complexed with at least one iigand having a nitrogen-containing heterocycle,
such as 2,2'-bipyridine, 1 ,10-phenanthroline, or a derivative thereof, preferably a mono-, di-, or polyaJkoxy deriv-
45 ative of 2,2'-bipyridine or 1,10-phenanthroHne, and more preferably comprises 4,4 , -dialkoxy-2,2 , -bipyridine or
4,7-dialkoxy-1 ,1 0-phenanthroline, wherein the carbon to oxygen ratio of the aikoxy functional groups is sufficient
to retain solubility of the transition metal complex in water prior to crosslinking.
47. The method of claim 46, wherein the nitrogen-containing heterocycle comprises 4,4'-dimethoxy-2,2'-bipyridine or
so 4,7-dimethoxy-1 ,10-phenanthroline.
48. The method of claim 46, wherein the osmium complex comprises osmium complexed with a polymeric Iigand,
wherein the .polymeric Iigand comprises a nitrogen-containing heterocycle.
55 49. The method of claim 48, wherein the polymer comprises poly(4-vinyl pyridine) or poly (1 -vinyl imidazole).
50. The method of claim 49, wherein the redox mediator comprises Os[4,4 , -dimethoxy-2 ) 2 , -bipyridine] 2 CI +/+2 or Os
[4,7-dimethoxy-1,10-phenanthroline] 2 CI +/+2 complexed with poly(1-vinyl imidazole).
24
EP 0 958 495 B1
51. The sensor of claim 7, wherein a portion of the air-pxidizable redox mediator is oxidized when packaging the
analytical sensor in an atmosphere containing molecular oxygen.
52. The method of claim 50, wherein greater than 90% of the redox mediator is in an oxidized state after being stored
5 for more than one month.
53. An analyte measurement system comprising:
a. a sensor of any of the claims 1 -23, 40, and 51 ; and
>0 b. a coulometer operatively connected to the sensor and configured to measure the cumulative electrical
charge flowing through the sensor.
PatentansprQche
15
1. Elektrochemischer Sensor zur Bestimmung der Konzentration eines Analyten in einer Probe, wobei der Sensor
aufweist:
wenigstens eine Arbeitselektrode (22);
20 wenigstens eine Gegenelektrode (24);
wobei die Elektroden, im Gebrauch, mit einer externen Elektronik einer MeBvorrichtung verbunden sind;
wenigstens eine Probenkammer (26), wobei die wenigstens eine Probenkammer
25 (i) eine Probenkammer ist, die die Probe in elektrolytischem Kontakt mit der Arbeitselektrode halt und so
bemessen ist, da(3 sie nicht mehr als etwa 1 |xl der Probe enthalt; oder
(ii) eine Probenkammer ist, die wenigstens eine MeBzone aufweist, die so bemessen ist, da(3 sie nicht mehr
als etwa 1pJ Probe enthalt, wobei die MeBzone auf wenigstens zwei Seiten von der Arbeits- und der Gegen-
elektrode begrenzt ist;
30
wobei der Sensor einen nicht auslaugbaren Redoxmediator auf der Arbeitselektrode enthalt; und
wobei der Sensor gegebenenfalls ein Sorbensmaterial (34) enthalt, das wenigstens teilweise innerhalb der
Probenkammer oder wenigstens teilweise innerhalb der MeBzone angeordnet ist.
35 2. Sensor nach Anspruch 1 , bei dem die Arbeitselektrode und die Gegenelektrode Metall oder Kohlenstoff umfassen,
und der Sensor den nicht auslaugbaren Redoxmediator auf der Arbeitselektrode enthalt.
3. Sensor nach Anspruch 2, wobei der Sensor auBerdem ein zweites Elektronenubertragungsmittel, vorzugsweise
ein Enzym, wie beispielsweise Glucoseoxidase, auf der Arbeitselektrode aufweist, wobei das zweite Obertragungs-
40 mittel gegebenenfalls auf der Arbeitselektrode immobilisiert ist und/oder gegebenenfalls nicht auslaugbar ist.
4. Sensor nach Anspruch 1 , wobei die Arbeitselektrode und die Gegenelektrode Metall oder Kohlenstoff umfassen,
und der Sensor das Sorbensmaterial einschlieBt, das wenigstens teilweise innerhalb der Probenkammer oder
wenigstens teilweise innerhalb der MeBzone angeordnet ist.
45
5. Sensor nach irgendeinem der Anspruche 1 bis 4, wobei die Probenkammer oder die MeBzone so bemessen ist,
daB sie nicht mehr als etwa 0,5 uJ, vorzugsweise nicht mehr als etwa 0,2 uJ, vorzugsweise nicht mehr als etwa 0,1
uJ der Probe enthalt.
50 6. Sensor nach irgendeinem der Anspruche 1 bis 5, wobei der Redoxmediator auf der Arbeitselektrode immobilisiert
ist.
7. Sensor nach irgendeinem der Anspruche 1 bis 6, wobei der Redoxmediator ein luftoxidierbarer Redoxmediator ist,
wobei vorzugsweise wenigstens 90% des luftoxidierbaren Redoxmediators vor der Einfuhrung der Probe in einem
55 oxidierten Zustand voriiegt.
8. Sensor nach irgendeinem der Anspruche 1 bis 7, wobei der Redoxmediator einen Ubergangsmetallkomplex um-
faBt, wie beispielsweise einen Osmium-, Ruthenium-, Eisen- Oder Cobaltkomplex, und vorzugsweise Osmium ist,
/
25
EP0 958 495 B1
9.
das mft wenigstens einem Liganden mit einem stickstoffhaltigen Helerocyclus komplexiert ist, wie beispielsweise
mit 2,2-Bipyridin, 4,4'-Dimethyl-2 l 2*-bfpyridin ( 4,4'-Dialkoxy-2 1 2'-bipyridin, 1,10-PhenanthroIin, 4,7-Dimethyl-
1,10-pnenanthrolin, 4,7-Dialkoxy-1,10-phenanthrolin oder Derivaten davon.
Sensor nach Anspruch 8, wobei der Ligand mit einem stickstoffhaltigen Heterocyclus 2,2'-Bipyridin 1 1 0-phenan-
throlin oder em Derivat davon umfaBt, wie beispielsweise ein Mono-, Di- oder Polyalkoxyderivat von 2 2'-Bipyridin
° 6 V ^°-^ enanthrolin ' w „ obel da * Verhaltnis von Kohlenstoff zu Sauerstoff der funktionellen Alkoxygruppe aus-
reicht, die Loslichkeit des Ubergangsmetallkomplexes in Wasser vor dem Vemetzen zu gewahrleisten.
10. Sensor nach Anspruch 8 oder 9, wobei das Verhaltnis von Kohlenstoff zu Sauerstoff der funktionellen Alkoxygrup-
pen ausreicht, die Loslichkeit des Obergangsmetallkomplexes in Wasser vor dem Vernetzen zu gewahrleisten
11
Sensor nach irgendeinem der Anspruche 1 bis 10, wobei das Sorbensmaterial ein freies Volumen von etwa 5%
bis 50%, vorzugsweise von etwa 1 0% bis 25% des Gesamtvo lumens des Sorbensmaterials aufweist.
12. Sensor nach irgendeinem der Anspruche 1 bis 11, wobei der Sensor auBerdem eine oder mehrere zusatzliche
Arbeitselektroden aufweist.
13. Sensor nach irgendeinem der Anspruche 1 bis 12, wobei wenigstens eine Arbeitselektrode und wenigstens eine
Gegenelektrode wenigstens ein einander gegenuberliegendes Elektrodenpaar bilden, wobei gegebenenfalls jedes
Paar auf einen spezifischen Analyten anspricht.
14. Sensor nach Anspruch 13, wobei die Arbeitselektrode und die Gegenelektrode durch einen Abstand von nicht
mehr als etwa 0,2 mm, oder nicht mehr als etwa 0, 1 mm, oder nicht mehr als etwa 0,05 mm voneinander getrennt
sind.
15. Sensor nach Anspruch 13, wobei wenigstens ein Paar sich gegeniiberiiegender Elektroden eine Arbeitselektrode
umfaBt, die em nicht auslaugbares Enzym und einen Redoxmediator aufweist, und worin wenigstens ein anderes
Paar von sich gegeniiberliegenden Elektroden eine Arbeitselektrode umfaBt, die einen nicht auslaugbaren Re-
doxmediator in Abwesenheit des Enzyms entbalt.
16. Sensor nach Anspruch 1 5, der auBerdem ein drittes Elektrodenpaar (46) aufweist, bei dem auf der Arbeitselektrode
kein Redoxmediator oder Enzym vorhanden ist.
17. Sensor nach irgendeinem der Anspruche 1 bis 16, wobei die Arbeitselektrode einen ersten Arm (122) und die
Gegenelektrode einen zweiten Arm (124) aufweisen, wobei ein Teil des ersten Arms einen Abschnitt des zweiten
Arms uberlappt, wobei die MeBzone oder die Probenkammer einen Bereich (21) zwischen den uberlappenden
Abschnitten des ersteri Arms und des zweiten Arms umfassen.
18. Sensor nach Anspruch 17, wobei der erste Arm eine Extralange aufweist und der uberlappende Abschnitt des
ersten Arms eine Breite aufweist, wobei ein Verhaltnis der Extralange des ersten Arms zu der Breite des uberlap-
penden Abschnitts des ersten Arms im Bereich von 0,1:1 zu 50:1 , vorzugsweise von 1:1 bis 15:1 und besonders
bevorzugt von 4:1 bis 10:1 betragt.
19. Sensor nach Anspruch 18, wobei die ersten und zweiten Arme sich unter einem Winkel von mehr als 0 Grad
uberschneiden.
20. Sensor nach irgendeinem der Anspruche 1 bis 1 9, wobei das Sorbensmaterial ein pulverformiges Material umfaBt,
das auf der Arbeitselektrode angeordnet ist.
21 . Sensor nach Anspruch 1 3, wobei das wenigstens eine Paar sich gegeniiberiiegender Elektroden ein Baslsmaterial
(1 02) mrt emer Ausnehmung (1 04) umfaBt und wenigstens eine von der Arbeitselektrode und der Gegenelektrode
in der Ausnehmung angeordnet ist.
22. Sensor nach irgendeinem der Anspruche 1 bis 21 , der auBerdem ein Fullstoff material aufweist, vorzugsweise ein
hydropses Fullstoff material, wobei das Fulistoffmaterial in der MeBzone oder in der Probenkammer angeordnet
ist, urn em Volumen der MeBzone oder der Probenkammer zu verringern, das fur die Probe zur Verfugung steht
26
EP 0 958 495 B1
23. Sensor nach den Anspruchen 1 bis 22, wobei der Sensor in einer Pufferlosung, die 10 mM Glucose enthalt, ein
Signal erzeugt, das wenigstens 9 mal groBer ist als ein Signal, das in einer Pufferlosung ohne Glucose erzeugtwird.
24. Verfahren zur Bestimmung einer Konzentration eines Analyten in einer Probe, das das Inkontaktbringen der Probe
mit dem elektrochemischen Sensor nach irgendeinem der Anspruche 1 bis 23 und die Bestimmung der Konzen-
tration des Analyten in der Probe mittels Coulometrie umfaBt.
25. Verfahren nach Anspruch 24, wobei die Stufe des Inkontaktbringens dadurch durchgefCihrt wird, daB man die
Probe mit dem Sorbensmaterial in Kontakt bringt, urn die Probe in die Probenkammeroder MeBzone einzusaugen.
26. Verfahren nach Anspruch 24, wobei die Probenkammer durch Kapillarwirkung gefullt wird,
27. Verfahren nach Anspruch 24, wobei der Schritt der Bestimmung der Konzentration des Analyten umfaBt:
is Elektrolysieren von wenigstens 90% des Analyten, der in einer MeBzone vorhanden ist, dadurch, daB man
ein Potential zwischen der Arbeits- und der Gegenelektrode anlegt, und zwar vorzugsweise in weniger ais
etwa 5 Minuten, und starker bevorzugt in weniger als etwa 1 Minute;
Bestimmen einer eiektrischen Ladung, die zur Elektrolyse des Analyten gebraucht wird; und
Korrelieren der eiektrischen Ladung mit der Konzentration des Analyten in der Probe,
w
20
25
30
55
wobei die Bestimmungsstufe gegebenenfalls mit den folgenden Stufen durchgefuhrt wird:
Messen eines Stroms, der an der Arbeitselektrode erzeugtwird, zu zwei oder mehrZeitpunkten, wahrend der
Analyt elektrolysiert wird; und
Integrieren der gemessenen Strome uber die Zeit, urn die eiektrische Ladung zuerhaiten, diefurdie Elektrolyse
des Analyten gebraucht wird.
28. Verfahren nach Anspruch 24, wobei die Stufe der Bestimmung der Konzentration des Analyten durch Coulometrie
die Schritte umfaBt:
Elektrolysieren eines Teils eines Analyten dadurch, daB man ein Potential an die Arbeitselektrode und Ge-
genelektrode anlegt;
Messen eines Stroms, der an der Arbeitselektrode erzeugt wird, zu zwei oder mehr Zeitpunkten wahrend der
Elektrolyse;
35 Extrapolieren einer Stromkurve auf der Basis der gemessenen Strome;
Integrieren der Stromkurve uber die Zeit, urn eine eiektrische Ladung zu erhaiten, die erforderlich ist, urn
wenigstens 90% des Analyten zu elektrolysieren; und
Korrelieren der eiektrischen Ladung mit der Konzentration des Analyten in der Probe.
40 29. Verfahren nach Anspruch 24, wobei der elektrochemlsche Sensor erste und zweite Elektrodenpaare aufweist,
wobei jedes Paar eine Arbeitselektrode umfaBt, wobei das erste der Elektrodenpaare einen nicht auslaugbaren
Redoxmediator und ein nicht auslaugbares Enzym auf der Arbeitselektrode aufweist, und wobei das zweite der
Elektrodenpaare einen nicht auslaugbaren Redoxmediator in Abwesenheit von Enzym auf der Arbeitselektrode
aufweist.
45
30. Verfahren nach Anspruch 29, wobei die Stufe der Bestimmung der Konzentration des AnaJyten die Schritte umfaBt:
im wesentlichen gieichzeitig, und zu zwei oder mehr Zeitpunkten, Messen eines ersten Stroms, der an dem
ersten Elektrodenpaar erzeugt wird, sowie eines zweiten Stroms, der an demzweiten Elektrodenpaar erzeugt
so wird;
Integrieren der gemessenen ersten Strome uber die Zeit, urn eine erste Ladung zu erhaiten;
Integrieren der gemessenen zweiten Strome uber die Zeit, um eine zweite Ladung zu erhaiten;
Abziehen der zweiten Ladung von der ersten Ladung, um eine rauschverminderte Ladung zu erhaiten; und
Korrelieren der Konzentration des Analyten mit der rauschverminderten Ladung.
31. Verfahren nach Anspruch 24, wobei das Verfahren, vor den Stufen des Inkontaktbringens und Bestimmens, die
weiteren Stufen umfaBt:
27
EP 0 958 495 B1
Bereitstellen des elektrochemischen Sensors nach irgehdeinem der Anspruche 1 bis 25, wobei der Sensor
eines oder mehrere Paare von sich gegenuberliegenden Elektroden aufweist, wobei jedes Eiektrodenpaar
eine Arbeitselektrode, eine Gegenelektrode und eine MeBzone zwischen der Arbeitselektrode und der Ge-
genelektrode aufweist,
5 . Messen einer Kapazitanz von wenigstens einem der Elektrodenpaare; und
Errechnen des Volumens der MeBzone aus der Kapazitanzmessung.
32. Verfahren nach Anspruch 24, wobei der Sensor einen Redoxmediator auf der Arbeitselektrode aufweist, und wobei
die molare Menge des Redoxmediators in einer reduzierten Form vor der Einfuhrung der Probe geringer ist als,
10 auf stochiometrischer Basis, 5% der erwarteten molaren Menge des Analyten, der elektrolysiert werden soil, und
wobei die Stufe der Bestimmung der Konzentration des Analyten umfaBt:
Elektrolysieren von weniger als etwa 1 uJ der Probe.
is 33. Verfahren nach Anspruch 32, wobei der Sensor wenigstens zwei Arbeitselektrode n aufweist, wobei jede Arbeits-
elektrode eine zugeordnete MeBzone aufweist, und wobei die Stufe der Bestimmung der Konzentration des Ana-
lyten das Mitteln von Messungen von den wenigstens zwei Arbeitselektroden umfaBt, und gegegebenfalls auBer-
dem die Eliminierung von Messungen umfaBt, die einen Schwellenwert uberschreiten, und gegebenenfalls auBer-
dem das erneute Mitteln der Messungen ohne die eliminierten Messungen.
20
34. Ein AnalytenmeBsystem, das umfaBt:
eine Probengewinnungseinrichtung, vorzugsweise einTeil (54) zum Anstechen der Haut, wie beispielsweise
eine Lanzette, urn eine Patientenprobe zu erzeugen;
25 den elektrochemischen Sensor nach irgendeinem der Anspruche 1 bis 23; und
gegebenenfalls eine Transporteinrichtung, wie beispielsweise ein saugendes Material, eine Kapiilarkammer,
ein Sorbensmaterial oder eine Pumpe, urn die Probe, die durch die Probengewinnungseinrichtung erzeugt
wurde, in die Probenkammer oder die MeBzone zu transportieren.
30 35. System nach Anspruch 34, wobei die Einrichtung zum Anstechen der Haut ein Teil des Sensors ist.
36. Verfahren zur Messung eines Analyten in einer Patientenprobe, wobei das Verfahren umfaBt:
inkontaktbringen des Patienten mit einem AnalytenmeBsystem, wobei das System umfaBt:
35
eine Probengewinnungseinrichtung zur Erzeugung einer Patientenprobe; und
den elektrochemischen Sensor nach irgendeinem der Anspruche 1 bis 23, urn den Analyten in der Probe
zu messen,
40 Gewinnen einer Probe unter Verwendung der Probengewinnungseinrichtung;
Transportieren eines Teils der Probe zu der MeBzone oder der Probenkammer des elektrochemischen Sen-
sors, wobei die Stufe des Transportierens gegebenenfalls das Einsaugen der Probe in die MeBzone oder die
Probenkammer unter Verwendung des Sorbensmaterials umfaBt; und
Bestimmen der Konzentration des Analyten in der Probe durch Coulometrie.
45
37. Verfahren nach Anspruch 36, wobei die Probengewinnungseinrichtung ein Teil des Sensors ist.
38. Verfahren nach den Anspruchen 36 oder 37, wobei die Probengewinnungseinrichtung ein Element zum Anstechen
der Haut, wie beispielsweise eine Lanzette umfaBt, und der Schritt der Gewinnung einer Probe das Anstechen
so der Haut des Patienten an einem Ort, der nicht ein Finger des Patienten ist, umfaBt, urn eine Probe zu erzeugen.
39. Verfahren zur Bestimmung einer Konzentration eines Analyten in einer Probe, wobei das Verfahren die Schritte
umfaBt:
55 Verblnden der Elektroden eines elektrochemischen Sensors, der eine Arbeitselektrode, eine Gegenelektrode
und eine Probenkammer aufweist, urn die Probe in einem elektrolytischen Kontakt mit der Arbeitselektrode
und der Gegenelektrode zu halten, mit der externen Elektronik einer MeBvorrichtung;
Inkontaktbringen der Probe mit dem Sensor;
28
EP 0 958 495 B1
Halten der Probe in einer stromungsfreien Weise innertialb der Probenkammer;
Anlegen eines Potentials zwischen der Arbeits- und der Gegenelektrode, urn den Analyten in einem Tei! der
Probe innerhalb der Probenkammer zu elektrolysieren, wobei derTeil der Probe, in der der Anatyt elektrolysiert
wird, ein Volumen von weniger als 1 \i\ aufweist;
5 Messen des Stroms, der durch den elektrochemischen Sensor erzeugt wird, zu einer Vielzahl von Zertpunkten,
wahrend der Analyt in dem Teil der Probe elektrolysiert wird; und
Bestimmen, durch Coulometrie, einer Konzentration des Analyten in der Probe, unter Verwendung der ge-
messenen Strome.
10 40. Sensor nach Anspruch 7, wobei der Sensor auBerdem ein zweites Elektronenubertragungsmittel aufweist, wie
beispielsweise ein Enzym, das auf dem Trager aufgetragen ist und mit dem Redoxmediator in Kontakt ist, wobei
das zweite Elektronenubertragungsmittel gegebenenfalls auf dem Trager immobilisiert ist.
15
41. Verfahren zur Bestimmung einer Konzentration eines Analyten in einer Probe, das die Schritte umfaBt:
Inkontaktbringen der Probe mit dem Sensor nach Anspruch 7 oder Anspruch 40, und
Korrelieren der Konzentration des Analyten in der Probe mit einer Veranderung des Oxidationszustands des
Redoxmediators in Gegenwart von Analyten.
20 42. Verfahren nach Anspruch 41, wobei der Sensor einen optischen Sensor umfaBt, und die Stufe des Korrelierens
der Konzentration des Analyten umfaBt:
Bestrahlen des Redoxmediators mit Licht;
Messen der Reaktion des Redoxmediators auf die Bestrahlung mit Licht; und
25 Korrelieren der Konzentration des Anaiyten mit der gemessenen Antwort des Redoxmediators.
43. Verfahren nach Anspruch 42, wobei die Stufe der Messung der Antwort des Redoxmediators auf die Bestrahlung.
mit Licht das Messen der Absorption oder des Transmissionsgrads des eingestrahlten Lichts durch den Redox-
mediator umfaBt.
30
44. Verfahren nach Anspruch 42, wobei der Schritt der Messung der Antwort des Redoxmediators auf Bestrahlung
mit Licht das Messen der Fluoreszenz des Redoxmediators nach der Bestrahlung mit Licht, oder die Messung der
Reflektion von Licht durch den Redoxmediator umfaBt.
35 45. Verfahren nach Anspruch 41 , wobei der Sensor einen elektrochemischen Sensor umfaBt, und der Schritt der Kor-
relation der Konzentration des Analyten umfaBt:
Anlegen eines eiektrischen Potentials an den Redoxmediator;
Messen eines Stroms in einem oder mehreren Intervallen, wobei der Strom als Reaktion auf die Elektrofyse
40 des Redoxmediators in Gegenwart des Analyten erzeugt wird; und
Korrelieren der Konzentration des Analyten mit dem gemessenen Strom.
46. Verfahren nach Anspruch 41, wobei der Redoxmediator einen Obergangsmetallkomplex umfaBt, wie beispiels-
weise einen Osmiumkomplex, und vorzugsweise Osmium, das mit wenigstens einem Liganden mit einem stick-
45 stoffhaltigen Heterocyclus komplexiert ist, wie beispielsweise mit 2,2'-Bipyridin, 1 ,10-Phenanthrolin oder einem
Derivatdavon, vorzugsweise einem Mono-, Di- oder Polyalkoxyderivat von 2,2'-Bipyridin oder 1 ,10-Phenanthrolin,
und noch starker bevorzugt mit 4,4 , -Dialkoxy-2,2 , -bipyridin oder 4,7-Dialkoxy-1,10-phenanthrolin, wobei das Ver-
haltnis von Kohlenstoff zu Sauerstoff der funktionalen Aikoxygruppen ausreicht, urn die Loslichkeit des Ubergangs-
metallkomplexes in Wasser vor dem Vernetzen zu gewahrleisten.
50
47. Verfahren nach Anspruch 46, wobei der stickstoffhaltige Heterocyclus 4 ( 4'-Dimethoxy-2,2 , -bipyridin oder 4,7-Di-
methoxy-1,10-phenanthrolin umfaBt.
48. Verfahren nach Anspruch 46, wobei der Osmiumkomplex mit einem pofymeren Liganden komplexiertes Osmium
55 umfaBt, wobei der polymere Ligand einen stickstoffhaltigen Heterocyclus umfaBt.
49. Verfahren nach Anspruch 48, wobei das Polymer Poly(4-vinylpyridin) oder Poly(1 -vinylimidazolin) umfaBt.
29
EP 0 958 495 B1
50. Verfahren nach Anspruch 49, wobei der Redoxmediator Os [4,4'-Dimethoxy-2 2'-biDvridinUCK+2 odf »r rw 7 rv
methoxy-UO-phenantbrolinfe^^ Oder Os[4,7-D,-
51. Sensor nach Anspruch 7, wobei ein Teil des luftoxidieroaren Redoxmediators oxidiert wird wenn der annMhrh.
Sensor .n einer Atmosphare verpackt wird, die molekularen Sauerstoffenlhalt. analytische
52. Verfahren nach Anspruch 50, wobei sich mehr als 90% des Redoxmediators nach einer Laoeruna fur mehr «k
einen Monat in einem oxidierten Zustand befinden. Lagerung fur mehr als
53. AnalytenmeBsystem, das umfaBt:
a. einen Sensor nach einem der Anspruche 1 -23, 40 und 51 ; und
b ein Coulometer, das funktionell mil dem Sensor vetbunden ist und so ausgelegt ist, dass es die kumulative
elektrische Ladung miBt, die durch den Sensor flieSt. xumuiatrve
Revendications
1 ' c^w teCtr ° Chim,qUe P ° Ur d6,eminer ' a — " *» anafyte dans un achantBlon, le detecteur
au moins une electrode de travail (22) ;
au moins une electrode auxiliaire (24) ;
lesquelles electrodes, en utilisation, so'nt raccordees a I'electronique exteme d'un instrument de mesure •
au moms une chambre a echantillon (26). dans lequel .adite chambre a 6chamillon est "
SZf^ZS? T nti " 0n P ° Ur maint6nir |,6chantillon e " electrolytlque avec I'electrode de
ravail et dlmenslonnee pour contenir un maximum de 1 ul d'echantillon environ ou
(11) une chambre a echantillon comprenant au moins une zone de mesure, dimensionnee pour contenir
i 1 h ' VT m T n environi dans ,aquel,e ,a zone de - w^-SSISS
cotes par I electrode de travail et l'6lectrode auxiliaire ;
ledit detecteur contenant un mediateur d'oxydoreduction non lixiviable sur I'electrode de travail ■ et
edit detecteur contenant facultativement un sorbant (34) dispose au moins partiellement a Pin'terieur de la
chambre a echantillon ou au moins partiellement a rinterieur de la zone de mesure
2 ' ^Xit^JI^X V d3nS ' eqUel '' 6leC,r0de * traVaH 6t WeM auxi,iaire sont de
S5. ' 6teCleUr ° 0mprend ,e . m6diate " d'oxydoreduction non lixiviable sur I'electrode de
3 ' SSrontaua da or^f** 0 " *' *"* ' eqUe ' '* det6CteUr COmprend en ° utre u " second a 9^t de transfert
To , h P , f enZyme te " e que ,a glucose ox y dase ' sur de travail, dans lequel le
HxTvSe 9 nSfert ,9CUftatiVement "™** a « .'electrode de travel, et/ou est facuitativemS non
detecteur comprend un sorbant dispose au moins partiellement a I'interieur de la cham-
bre a echantillon ou au moms partiellement a I'interieur de la zone de mesure.
5 " m—rl^nT qUe ' COnqUe ^ revendications 1 a 4 ' da "* M"* 'a chambre a echantillon. ou .a zone de
Z X d? D °Z=„ P ° Ur C ° n, ! nir Un maXimiJm de °' 5 ^ environ ' de V* 6 ™™ «n maxtaum de 0,2 J
environ, de preference un maximum de 0,1 H l environ d'echantillon.
6 ' SSSSr ^ reV6ndiCati0nS 1 8 5 ' d8nS leqUel * ^ iateUr d ^ d ^n est immo-
? ' n^ZT^Z quelcon( > ue des revendications 1 a 6, dans lequel le mediateur d'oxydoreduction est un
med,ateur d oxydoreduction oxydable a .'air, avec de preference au moins 90 % du mediateur d'o ^ucHon
30
EP 0 958 495 B1
oxydable a I'air a I'etat oxyde avant ^introduction de I'echantillon.
8. Detecteur selon Tune quelconque des revendications 1 a 7, dans lequel le mediateur d'oxydoreductlon comprend
un complexe metallique de transition, tel qu'un complexe d'osmium, de ruthenium, de fer ou de cobalt, et de
preference un complexe d'osmium avec au moins un ligand ayant un compose heterocyclique azote, tel que la
bipyridine-2,2', le dimethyl-4,4'bipyridine-2,2\ !e dialkoxy-4,4' bipyridine-2,2*, la phenanthroline-1,10, le dimethyl-
4,7 phenanthroline-1,10, le dialkoxy-4,7 phenanthroline-1 ,10, ou les derives de ceux-ci.
9. Detecteur selon la revendication 8, dans lequel le ligand ayant un compose heterocyclique azote comprend de la
bipyridine-2,2', de la phenanthroline-1,10, ou un derive de celles-ci, tel qu'un derive mono-, di- ou polyalkolxy de
la bipyridine-2,2' ou de la phenanthroline-1,10, dans lequel le rapport carbone sur oxygene des groupements
fonctionnels aikoxy est suffisant pour conserver la solubiiite du complexe metallique de transition dans I'eau avant
la reticulation.
10. Detecteur selon la revendication B ou 9, dans lequel le rapport carbone sur oxygene des groupements fonctionnels
aikoxy est suffisant pour conserver la solubiiite du complexe metallique de transition dans I'eau avant la reticulation.
11. Detecteur selon Tune quelconque des revendications 1 a 10, dans lequel le sorbant a un volume mort de 5 % a
50 % environ, de preference de 1 0 % a 25 % environ du volume total du sorbant.
12. Detecteur selon Tune quelconque des revendications 1 a 11, dans lequel le detecteur comprend en outre une ou
plusieurs electrodes de travail supplementaires.
13. Detecteur selon Tune quelconque des revendications 1 a 12, dans lequel au moins une electrode de travail et au
moins une Electrode auxiliaire forment au moins une paire d'electrodes en regard, dans lequel facultativement
chaque paire reagit a un analyte specifique.
14. Detecteur selon la revendication 13, dans lequel la distance separant I'electrode de travail de I'electrode auxiliaire
n'excede pas 0,2 mm environ, ou 0,1 mm environ, ou 0,05 mm environ.
15. Detecteur selon la revendication 13, dans lequel au moins une paire d'electrodes en regard est composee d'une
electrode de travail comprenant un mediateur d'oxydoreductlon et une enzyme non iixiviables, et dans lequel au
moins une autre paire d'electrodes en regard est composee d'une electrode de travail comprenant un mediateur
d'oxydoreductlon non Itxiviable en I'absence de Tenzyme.
16. Detecteur selon la revendication 1 5, comprenant en outre une troisieme paire d'electrodes (46) n'ayant ni mediateur
d'oxydoreduction ni enzyme sur I'electrode de travail.
17. Detecteur selon Tune quelconque des revendications 1 a 16, dans lequel I'electrode de travail comprend un premier
bras (122) et I'electrode auxiliaire comprend un second bras (124), une partie du premier bras chevauchant une
partie du second bras, dans lequel la zone de mesure, ou la chambre a echantillon, comprend une region (21)
entre les parties chevauch antes du premier et du second bras.
18. Detecteur selon la revendication 17, dans lequel le premier bras a une longueur supplemental et la partie che-
vauch ante du premier bras a une largeur, dans lequel le rapport de la longueur supplemental du premier bras
sur la largeur de la partie chevauchante du premier bras varie de 0,1/1 a 50/1 , de preference de 1/1 a 1 5/1 , et de
maniere preferee entre toutes de 4/1 a 10/1 .
19. Detecteur selon la revendication 1 8, dans lequel le premier et le second bras se coupent a un angle superieur a
0 degre.
20. Detecteur selon Tune quelconque des revendications 1 a 19, dans lequel le sorbant comporte un materiau en
poudre dispose sur ['electrode de travail.
21. Detecteur selon la revendication 13, dans lequel ladite paire d'electrodes en regard comprend un materiau de
base (102) ayant une encoche (104) et au moins une de Pelectrode de travail et de I'electrode auxiliaire disposee
dans Tencoche.
31
EP 0 958 495 B1
22 Detecteur selon rune quelconque des revendications 1 a 21, comprenant en outre un materiau de rempKssage
de Serene u matiiau de remplissage hydrophile. dans lequel le materiau de remplissage est d,spos6 dans
fa zone de mesurTou dans la chambre a echantillon, pour reduire un volume de la zone de mesure ou de la
chambre a echantillon disponible pour I'echantillon.
23 Detecteur selon I'une queloonque des revendications 1 a 22, dans lequel le detecteur produit un signal dans una
2 \ ol u«
tampon sans glucose.
24. Precede pour determiner une concentration tfun.ana.yte dans un echantillon. ^ contact de
echantillon avec le detecteur electrochimique selon I'une quelconque des revendications 1 a 23. et la determina
tion de la concentration de I'analyte dans I'echantillon par coulometne.
25 Precede selon la revendication 24. dans lequel I'etape de mise en contact est reaHsee en ^ «n co ntact
Uchantillon avec ie sorbant pour introduire par effetdemecherechantillon dans la chambre a echantrilonou dans
la zone de mesure.
2$. Precede selon la revendication 24, dans lequel la chambre a echantillon se remplit par capillarite.
27. Precede selon la revendication 24, dans .equel I'etape de determination de la concentration de I'analyte comporte
les phases suivantes :
electrolyse d'au moins 90 % de I'analyte present dans la zone de mesure par application tfun potentiel a
^S!S^!^ travail et .'electrode auxiliaire, de preference en moins de 5 minutes environ, et de ma-
niere pref6ree entre toutes en moins de 1 minute environ ;
determination d'une charge electrique utilisee pour S^ 0 '^/ 3 ^ e . ; ane . 1(SehanHllon .
et correlation entre la charge electrique et la concentration de I'analyte dans I echantillon
daSCei fSe de determination est facultativement rea.isee selon les phases suivantes :
mesure d'un courant genere au niveau de I'electrode de travail a deux instants ou plus pendant Electrolyse
iteg*^
I'analyte.
28. Precede selon la revendication 24. dans lequel I'etape de determination de la concentration de I'analyte par cou-
lometrie comporte les phases suivantes :
electrolyse d'une partie de I'analyte par application d'un potentiel a travers I'electrode de travel, et I'electrode
melure tf un courant genere au nfceau de I'electrode de travail a deux instants ou plus pendant .'electrolyse ;
extraDolation d'une courbe de courant basee sur les courants mesurds ;
SSSSm 1 temps de la courbe de courant pour obtenir une charge e.ectnque necessa,re pour elec-
trolyserau moins 90 %de I'analyte; M-h»nHiinn
et correlation entre la charge electrique et la concentration de I'analyte dans 1 echantillon.
29. Precede selon la revendication 24, dans .equel ie detecteur electrochimique est ^^^^Sl
seconde paire d'electrodes, cheque paire comportant »" e
Tabsence d'enzyme.
30. Precede selon la revendication 29. dans lequel I'etape de determination de la concentration de I'analyte comporte
les phases suivantes :
mesure pratiquement simultanee. et a deux instants ou plus, tfun premier courant 0**"">*ȣ de la
premiere paire d'electrodes et tfun second courant genere au nrveau de la s^J™"™"*™ 6 ** '
integration dans le temps des premiers courants mesures pour obtenir une seconde charge
soustraction de la seconde charge de la premiere charge pour obtenir une charge * brurt redu.t ,
32
EP 0 958 495 B1
et correlation entre la concentration de I'analyte et la charge a bruit reduit.
31 . Precede selon la revendication 24, le procede cqmportant, avant les etapes de mise en contact et de determination,
les autres phases suivantes :
5
fourniture du detecteur electrochimique selon Tune quelconque des revendication s 1 a 23, dans lequel le
detecteur a une ou plusieurs paires d'electrodes en regard, chaque paire d'electrodes etant composee d'une
electrode de travail, d'une electrode auxiliaire et d'une zone de mesure entre Pelectrode de travail et Pelectrode
auxiliaire ;
to mesure d'une capacite d'au moins une des paires d'electrodes ;
et calcul du volume de la zone de mesure a partir de la mesure de capacite.
32. Procede selon la revendication 24, dans lequel le detecteur comprend un mediateur d'oxydoreduction sur I'elec-
trode de travail, et la quantite molaire de mediateur d'oxydoreduction sous forme reduite avant I'introduction de
is I'echantillon est inferieure, sur une base stoechiometrique, a 5 % de la quantite molaire theorique de I'analyte a
electrolyser ; et dans lequel I'etape de determination de la concentration de I'analyte comporte :
electrolyse de moins d'1 u.l d'echantillon.
20 33. Procede selon la revendication 32, dans lequel le detecteur comprend au moins deux electrodes de travail, chaque
electrode de travail ayant une zone de mesure associee, et dans lequel I'etape de determination de la concentration
de I'analyte comporte le calcul de la moyenne des mesures realisees sur lesdites electrodes de travail, et facul-
tativement comporte en outre PeJimlnation des mesures excedant une valeur-seuil, et facultativement comporte
en outre le recalcul de la moyenne des mesures sans les mesures eliminees.
25
34. Dispositif de mesure d'anatyte comprenant :
moyen d'acquisition d'echantillons, de preference un membre pour inciser la peau (54), tel qu'une lancette,
pour produire un echantillon d'un patient
30 le detecteur electrochimique selon I'une quelconque des revendications 1 a 23 ;
et facultativement un moyen de transport, tel un materiau a effet de meche, une chambre capillaire, un sorbant
ou une pompe, pour transporter I'echantillon produit par le moyen d'acquisition d'echantillons vers la chambre
a echantillon ou vers la zone de mesure.
35 35. Dispositif selon la revendication 34, dans lequel le membre pour inciser la peau fait partie integrante du detecteur.
36. Procede pour mesurer I'analyte dans un echantillon de patient, le procede comprenant :
mise en contact du patient avec le dispositif de mesure d'analyte, le dispositif comprenant :
40
un moyen d'acquisition d'echantillon pour produire un echantillon de patient ;
et le detecteur electrochimique selon I'une quelconque des revendications 1 a 23, pour mesurer I'analyte
dans I'echantillon ;
acquisition d'un echantillon avec le moyen d'acquisition d'echantillon ;
45 transport d'une partie de I'echantillon vers la zone de mesure ou la chambre a echantillon du detecteur
electrochimique, dans lequel I'etape de transport comprend facultativement I'introduction par effet de me-
che de I'echantillon dans la zone de mesure ou dans la chambre a echantillon a I'aide du sorbant ;
et la determination de la concentration de I'analyte dans I'echantillon par coulometrie.
50 37. Procede selon la revendication 36, dans lequel le moyen d'acquisition d'echantillon fait partie integrante du de-
tecteur.
38. Proc6de selon les revendications 36 et37, dans lequel le moyen d'acquisition d'echantillon comprend un membre
pour inciser la peau, tel une lancette, et I'etape d'acquisition d'echantillon comporte Pincision de la peau du patient
55 a un endroit autre que le doigt pour produire un echantillon.
39. Procede pour determiner une concentration d'un anaJyte dans un echantillon, le procede comportant les etapes
survantes :
33
20
25
40
45
50
55
EP0958495B1
raccordement des electrodes d'un detecteur electrochimique compose d'une electrode de travail, d'une elec-
3£SS!!; ? T^ c T, bre * * chanti,,on pour maintenir r6ohM » e * ~ £
I electrode de trava.l et ('electrode auxiliaire, a I'electronique externe d'un appareil de mesure •
mise en contact de I'echantillon et du detecteur ;
5 maintien de I'echantillon a I'interieur de la chambre a echantillon sans circulation ■
application d'un potentiel entre I'electrode de travail et I'electrode auxiliaire pour'electrolyser I'analyte dans
une^
lequel I'analyte est electrolyse a un volume inferieur a 1 uJ • «w«nimon aans
kishs e^otr r 6lec,rochimique * p,usieurs ins,an,s pendant que rana,yte —
et determination par coulometrie d'une concentration de I'analyte dans I'echantillon a I'aide des courants me-
40. Detecteur selon la revendication 7, dans lequel le detecteur comprend en outre un second agent de transfer!
elecfromque, tel qu'une enzyme, enduft sur le support et en contact avec le mediateur cfoSSSSTSS
lequel le second agent de transfert electronique est facultativement immobilise sur le support
41 . Precede pour determiner une concentration d'un analyte dans un echantillon, comportant les etapes suivantes :
mise en contact de I'echantillon avec le detecteur selon la revendication 7 ou la revendication 40 •
^Z^^X™«2ZT S ,,echanti,,on et un ch ' angement d ' e,at d ^ dation du
irradiation du m6diateur d'oxydoreduction avec de la lumiere ;
mesure de la reponse du mediateur d'oxydoreduction a I'irrad'iation par la lumi&re ■
et correlation entre la concentration de I'analyte et la reponse mesuree du mediateur d'oxydoreduction.
I l2t,i e 'n nir , re r endfcati ° n 42> danS ' eqUel '' 6,ape de meSure de la r6 P° nse du me diateur d'oxydoreduction
^:::^^zr^ ,a mesure de |,absorp,ion ' ou ,a transmittance - de ,a *
44 ' iTrS^Zt ZTT"™ ^ ^ ^ ***** meSUre de ' a r6ponSe du mediateur d'oxydoreduction
frriSf ■ 6 ? 0mP ° rte ' a meSUre dS 19 du mediateur d'oxydation- reduction apres
.rrad.at.on par la lum.ere, ou la mesure de la reflexion de la lumiere par le mediateur d'oxydoreduction.
45 " tecantiT "J 3 : 6VendiCa,i0n 41 ' dans le 1 uel le detecteur comprenant un detecteur electrochimique, et I'etape
de correlation de la concentration de I'analyte comporte : w p
application d'un potentiel electrique k travers le mediateur d'oxydoreduction • *
USS^StH " n ° U P I USieUrS J nt , erVal,eS * ' e C ° Urant 6,ant 96nere e " re P onse a 'Electrolyse du me-
aiateur d oxydoreduction en presence de I'analyte ;
et correlation entre la concentration de I'analyte et le courant mesure.
46 ' SI^h e '°? I 3 r r endiCat ! on 41 ' dans ^ le ^diateur d'oxydoreduction comprend un complexe metallique
un compose het6rocycl,que azote, tel que la bipyridine-2,2-, la phenanthroline-1 ,1 0, ou un derive de celles J de
ZTZZr TT m ° n0 '' ° U P °' yalk0lXy de ' a bi PV ridine -2.2' <>« de la phenanthroline-1,10, et de manure
ZTk calTn IT C ° mprend f dia ' kOX y- 4 ' 4, b'Pyrtdine-2,2' ou du dialkoxy-4.7 phenanthro.ine-1,10, dans Z
drcomXrmS.r. s rtir^
ou complexe metallique de transition dans I'eau avant la reticulation.
47. Precede seton la revendication 46, dans lequel le compose heterocyclique azote comprend du dimethoxv-4 4'
bipyndine^ ou du dimethoxy-4,7 phenanthroline-1 ,10. aimetnoxy 4,4
48. Precede selon la revendication 46, dans lequel le complexe d'osmium comprend de I'osmium en complexe avec
30
34
EP 0 958 495 B1
un ligand polymerique, dans lequel le ligand polymerique comprend un compose heterocyclique azote\ .
49. Procede selon la revendication 48, dans lequel le polymere comprend du po!y(vinyle pyridine-4) ou du poly(vinyle
imidazole-1).
50. Procede selon la revendication 49, dans lequel le mSdiateur d'oxydoreduction comprend de I'OsCdim^thoxy^^'
bipyridine^^^CI^ 2 ou de l'Os(dimethoxy-4,7 phenanthroline-1 ,1 0) 2 CI +/+2 en complexe avec du poly(vinyle imi-
dazole^).
51. Detecteur selon la revendication 7, dans lequel une partie du mediateur d'oxydoreduction oxydable a Pair est
oxydee au moment de I'embaliage du detecteur analytique dans une atmosphere contenant de I'oxygene mole-
culaire.
52. Procede selon la revendication 50, dans lequel plus de 90 % du mediateur d'oxydoreduction est a I'etat oxyde
apres une periode de stockage superieure a un mois.
53. Dispositif de mesure d'analyte comprenant :
un detecteur selon Tune quelconque des revendications 1 a 23, 40 et 51 ;
et un coulombmetre fonctionnellement raccorde au detecteur et configure pour mesurer la charge electrique
cumulee circulant dans le detecteur.
35
EP 0 958 495 B1
36
EP 0 958 495 B1
FIG. 2
30
37
EP 0 958 495 B1
EP 0 958 495 B1
EP 0 958 495 B1
FIG. 5
40
EP 0 958 495 B1
o
ti-
ro
CM
41
EP 0 958 495 B1
400
300-
200-
100-
• buffer
o serum
glucose (mM)
FIG. 7
42
EP 0 958 495 B1
EP 0 958 495 B1
FIG. 10
45
EP 0 958 495 B1
22
IS
1
24
FIG. 11A
124
222
FIG. 11C
46
EP 0 958 495 B1
FIG. 13B
FIG. 14B
47
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